Practical Aspects of Trapped Ion Mass Spectrometry, Volume V: Applications of Ion Trapping Devices (Modern Mass Spectrometry) [1 ed.] 1420083732, 9781420083736

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-8373-6 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Practical aspects of ion trap mass spectrometry / edited by Raymond E. March, John F.J. Todd. p. cm. -- (Modern mass spectrometry) Includes bibliographical references and index. ISBN 0-8493-4452-2 (vol. 1) 1. Mass spectrometry. I. March, Raymond E. II. Todd, John F.J. III. Series. QD96.M3P715 1995 539.7’.028’7--dc20 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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To ion trappers, young and old, everywhere.

Contents Preface.......................................................................................................................xi Volume IV Contents................................................................................................xxi Editors ....................................................................................................................xxv Contributors ..........................................................................................................xxix

PART I Ion Reactions Chapter 1

Ion/Ion Reactions in Electrodynamic Ion Traps ...................................3 Jian Liu and Scott A. McLuckey

Chapter 2

Gas-Phase Hydrogen/Deuterium Exchange in QuadrupoleIon Traps ............................................................................................. 35 Joseph E. Chipuk and Jennifer S. Brodbelt

Chapter 3

Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry in a Quadrupole Linear Ion Trap...................................... 59 Graeme C. McAlister and Joshua J. Coon

PART II Ion Conformation and Structure Chapter 4

Chemical Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural Characterization ....................... 83 Jennifer M. Froelich, Yali Lu, and Gavin E. Reid

Chapter 5

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in the Analysis of Peptides and Proteins .......................................... 121 Helen J. Cooper

Chapter 6

MS/MS Analysis of Peptide–Polyphenols Supramolecular Assemblies: Wine Astringency Approached by ESI-IT-MS ............ 153 Benoît Plet and Jean-Marie Schmitter

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Chapter 7

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Structure and Dynamics of Trapped Ions ......................................... 169 Joel H. Parks

Chapter 8

Applications of Traveling Wave Ion Mobility-Mass Spectrometry ....................................................................................205 Konstantinos Thalassinos and James H. Scrivens

PART III Ion Spectroscopy Chapter 9

The Spectroscopy of Ions Stored in Trapping Mass Spectrometers ................................................................................... 239 Matthew W. Forbes, Francis O. Talbot, and Rebecca A. Jockusch

Chapter 10

Sympathetically-Cooled Single Ion Mass Spectrometry ................ 291 Peter Frøhlich Staanum, Klaus Højbjerre, and Michael Drewsen

Chapter 11

Ion Trap: A Versatile Tool for the Atomic Clocks of the Future! ...................................................................... 327 Fernande Vedel

PART IV

Practical Applications

Chapter 12 Boundary-Activated Dissociations (BAD) in a Digital Ion Trap (DIT) ...................................................................................... 367 Francesco L. Brancia, Luca Raveane, Alberto Berton, and Pietro Traldi Chapter 13 The Study of Ion/Molecule Reactions at Ambient Pressure with Ion Mobility Spectrometry and Ion Mobility/ Mass Spectrometry ......................................................................... 387 Gary A. Eiceman and John A. Stone Chapter 14

The Role of Trapped Ion Mass Spectrometry for Imaging ............. 417 Timothy J. Garrett and Richard A. Yost

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Chapter 15

Technology Progress and Application in GC/MS and GC/MS/MS ................................................................ 439 Mingda Wang and John E. George III

Chapter 16 Remote Monitoring of Volatile Organic Compounds in Water by Membrane Inlet Mass Spectrometry ............................... 491 Romina Pozzi, Paola Bocchini, Francesca Pinelli, and Guido C. Galletti Author Index.........................................................................................................509 Subject Index ........................................................................................................ 513

Preface This monograph is Volume V of a miniseries devoted to (i) practical aspects of applications of mass spectrometry for the study of gaseous ions conined in ion traps, and (ii) treatments of the theory of ion coninement in each ion-trapping device. Volumes I–III were published in 1995 under the title Practical Aspects of Ion Trap Mass Spectrometry. Volume III, Chemical, Environmental and Biomedical Applications, is a companion to Volumes I and II, subtitled Fundamentals of Ion Trap Mass Spectrometry and Ion Trap Instrumentation, respectively. Volumes I–III are concerned principally with the history, theory, and applications of the quadrupole ion trap and, to a lesser degree, of the quadrupole mass ilter. Volume V, published in 2009 under the title Practical Aspects of Trapped Ion Mass Spectrometry, and subtitled Applications, is a companion to Volume IV, subtitled Theory and Instrumentation. The contents of Volume IV are given following the conclusion of this preface. The history of the quadrupole ion trap was presented in tabular form in Chapter 2 of Volume I as “The Ages of the Ion Trap” and, upon revisiting this table, one is struck by the spectacular progress that has been made in the ion-trapping ield since 1995. In the Preface to Volume II, we noted two exceptional landmarks in this history: irst, the invention of the quadrupole ion trap (and quadrupole mass ilter) by Wolfgang Paul and Hans Steinwedel, which was recognized by the award of the 1989 Nobel Prize in Physics, in part, to Wolfgang Paul and Hans Dehmelt; and, second, the discovery announced in 1983 of the mass-selective instability scan by George C. Stafford, Jr. On these two landmarks rested the entire ield of ion trap mass spectrometry. One of the table entries for 1990 was “Electrospray Ionization (Van Berkel, Glish, and McLuckey),” and Chapter 3 of Volume II was devoted to “Electrospray and the Quadrupole Ion Trap.” A further contribution, entitled “Electrospray/Ion Trap Mass Spectrometry – Applications,” by Hung-Yu Lin and Robert D. Voyksner, appeared as Chapter 14 in Volume III. The advent of electrospray ionization and its ready compatibility with ion-trapping devices has brought about a revolution in the accessibility of covalent compounds for examination by mass spectrometry in general and by quadrupole ion trap mass spectrometry in particular. For their development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules, John Fenn and Koicho Tanaka received the Nobel Prize in Chemistry for 2002. We add our congratulations and thanks to these Nobelists and to those from the mass spectrometry community. The enormous impact that electrospray ionization has made in biochemistry in general, and in the study of proteins in particular, is remarkable. Virtually every mass spectrometry laboratory is now equipped with electrospray ionization; compounds for which derivatization was previously essential for examination by electron impact can now be examined facilely in solution by direct infusion to an electrospray ionization source. As testament to this situation, more than half of the chapters presented in Volumes IV and V are concerned with the use of electrospray ionization. The practice of trapping gaseous ions and the applications thereof have expanded considerably during the past decade or so, in part due to the use of electrospray xi

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ionization but also as witnessed by the substantial growth in popularity of quadrupole ion traps and of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers, instruments that hitherto were regarded as being rivals rather than complementary technologies. In addition, we have seen the nascence of new methods for trapping ions, such as the Orbitrap™, the digital ion trap (DIT), the rectilinear ion trap (RIT), and the toroidal ion trap. Furthermore, during this period, there have been signiicant advances in the development and application of the quadrupole ion trap and of the quadrupole mass ilter, both standalone and in concatenation with other mass spectrometric instruments, for example, with Fourier transform ion cyclotron resonance and with time-of-light (TOF) mass spectrometers. New and/or modiied existing methods for ion processing have been developed and applied; these methods include electron capture dissociation (ECD), electron transfer dissociation (ETD), charge inversion, proton transfer reaction (PTR), electron transfer (ET), and ion attachment (IA). Other recent advances involving the coupling of ion mobility spectrometry (IMS) with mass spectrometry have brought about the introduction of high-ield asymmetric waveform ion mobility spectrometry (FAIMS) and traveling wave ion mobility mass spectrometry (TWIM-MS). Indeed, so many advances have occurred in the ion-trapping ield that we needed to consider a somewhat broader deinition of ion trapping compared with what has been employed hitherto; after several iterations, we arrived at the deinition proposed in Section 1.1 of Volume IV, “an ion is ‘trapped’ when its residence time within a deined spatial region exceeds that had the motion of the ion not been impeded in some way.” Clearly, this deinition includes those various forms of ion mobility spectrometry mentioned above. Armed with this deinition of ‘trapped ions,’ it seemed appropriate to the editors that a further volume in this mini-series could be undertaken, not limited to quadrupole devices but encompassing advances in all aspects of trapped ion mass spectrometry. When a commercial product has achieved a degree of market acceptance, which we believed was the case for the three volumes of Practical Aspects of Ion Trap Mass Spectrometry, one is reluctant to lose the connectivity within the miniseries upon embracing an expansion of the ield in question. Fortunately, a minor word change to Practical Aspects of Trapped Ion Mass Spectrometry saved the day. With this small but signiicant change in title, the expanded ield could be considered and included within the ‘practical aspects of ion trapping’ rubric. The collective response to our subsequent approaches to potential authors in the expanded ion-trapping ield was near overwhelming, so much so that in fact two monographs, Volumes IV and V, have resulted from this endeavor. Volume IV is entitled Theory and Instrumentation and is composed of six parts: Fundamentals, New Ion Trapping Techniques, Fourier Transform Mass Spectrometry, Quadrupole Rod Sets, 3D-Quadrupole Ion Trap Mass Spectrometry, and Photochemistry of Trapped Ions. Volume V is entitled Applications and features four parts: Ion Reactions, Ion Conformation and Structure, Ion Spectroscopy, and Practical Applications. Part 1. Ion Reactions is composed of three chapters in which ion reactions, that is, ion/neutral reactions or ion/ion reactions, are examined. Several ion-trapping devices have the capability for examining reactions of ions with neutral species and other ionic species where the extent of the reaction is monitored by the mass

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spectrometric function of the instrument. The quadrupole, or electrodynamic, ion trap is inherently compatible with the study of ion/ion reactions due to its unique ability to store simultaneously ions of both polarities in overlapping regions of space. In Chapter 1 is presented a review of the instrumental requirements for the study of ion/ion reactions. Particular emphasis is given to the use of an electrodynamic ion trap for the study of multiply-protonated peptide molecules with anions. The trapped ions assume characteristic sets of m/z-dependent frequencies of motion in the oscillating quadrupole ield of the ion trap, which allows ready manipulation of ions for ion isolation and activation, both of which are common elements in a tandem mass spectrometric experiment. The ‘tandem-in-time’ nature of the ion trap MSn experiment provides well-deined conditions for ion/ion reactions and permits determination of ion genealogy. A bath gas, such as helium at ca 1 mTorr, intended originally to cool the ions to the center of the trap so as to enhance both sensitivity and mass resolution upon mass analysis, improves ion/ion reaction eficiencies by maximizing the spatial overlap and minimizing the translational energies of the two ion clouds. Chapter 2 is focused on a particular type of ion/neutral reaction, namely that in which hydrogen atoms in the ion exchange with deuterium in the neutral reaction partner. The quadrupole ion trap mass spectrometer is well suited for investigations of such hydrogen/denterium (H/D) exchange reactions because the kinetics of reactions can be monitored accurately by varying the ion storage time. As illustrated in this chapter, applications of H/D exchange in quadrupole ion traps range from those involving small organic molecules, especially involving comparisons of isomers, to larger biological molecules for which conformational effects play a signiicant role. Chapter 3 considers the prospect of utilizing multiple ion-manipulation methodologies, which are available with ion trap mass spectrometers, to achieve whole protein sequence analysis; such analysis is described as top-down proteomics. The basis of this approach is the implementation of multi-functional tools for systematic ion manipulation and processing, where ion/ion reactions such as electron transfer, proton transfer, and ion attachment, represent one family of such tools. These technologies are inter-meshed with conventional ion trap processing methodologies of ion isolation and collision-induced dissociation. Concatenation of MSn scan functions from these individual components can constitute a versatile approach that promises to accelerate markedly the ield of large molecule mass spectrometry. The practical application of these processing methodologies in a linear ion trap requires modiication of the ion trap electronics to allow for the superimposition of a radiofrequency voltage on the end lenses, which allows for charge-sign independent trapping. Part 2. Ion Conformation and Structure presents discussions of structural characterization of proteins and peptides using quadrupole ion trap mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry, and the novel method known as traveling wave ion mobility mass spectrometry. In addition to the observation of collective luctuations of the molecular substructures within biomolecules, the organization of atoms in small ion clusters is investigated using electron diffraction. In Chapter 4 is discussed the ‘bottom-up’ or ‘shotgun’ tandem mass spectrometric approach to protein identiication and characterization, which is the complementary method to top-down proteomics that is discussed in Chapter 3. In order to overcome the limitations of bottom-up proteomics, chemical derivatization strategies are

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explored that direct the fragmentation of protonated peptides to the formation of sequence product ions. Such strategies can be employed to direct fragmentation to the formation of non-sequence product ions. Chapter 5 provides an overview of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and its applications in the structural characterization of peptides and proteins. The principles of FT-ICR, that is, ion motion, ion excitation/ detection, and instrumental considerations, are discussed and an explanation of the features of FT-ICR that make it so suitable for peptide/protein analysis is presented. New methods for the fragmentation of peptide and protein ions in FT-ICR mass spectrometry, such as sustained off-resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), and electron capture dissociation (ECD), are described in detail. Innovative hybrid FT-ICR instruments, which have recently become available, are reviewed. In conclusion, the chapter discusses the applications of FT-ICR in ‘bottom-up’ and ‘top-down’ proteomics. Chapter 6 is devoted to the tandem mass spectrometric investigation of supramolecular assemblies of peptides with non-covalently-bonded polyphenols. The quest of the speciic investigation recounted in this chapter was to gain insight at a molecular level into the interaction of polyphonies with proline-rich peptides, and to develop a future analytical methodology for the evaluation of astringency, speciically the astringency of wine. Two relevant points of interest are (i) polyproline peptides are subjects of intensive study, as is shown in the following chapter, Chapter 7, and (ii) the chemistry of proteins with non-covalently-bonded ligands is under examination because of the possibility of facile transport of ubiquitous compounds of doubtful environmental value into organs such as the liver. Analysis of these supramolecular assemblies of proline-rich peptides with a wide range of lavonoids (polyphenols), by means of energy-resolved mass spectrometry (ERMS), led to the creation of a relative afinity scale of the proline-rich peptides for the lavonoids examined. Chapter 7 describes quadrupole ion trap studies of the organization of atoms in small ion clusters and the observation of collective luctuations of the molecular substructures within biomolecules. The introduction of new ion sources, in particular metal-cluster aggregation sources and electrospray ionization, have provided unique opportunities to produce ion beams composed of metal atom clusters and biomolecules, respectively. Metal clusters are formed with a single charge but in a broad array of masses corresponding to the number of atoms, whereas biomolecular ions are generated for a single species in an ensemble of charge states. These studies take advantage of the advances in ion trap technology for lexible and reliable ion-cloud manipulation of higher mass ions required for the electron diffraction of, for example, Ag55+ and Au21−, and for luorescence measurements of dye-derivatized polyproline peptides. The results presented here enunciate clearly the ways in which these methods have contributed to our understanding of how the atoms are organized in small metal clusters and of the temperature dependence of local luctuations of biomolecular conformations. In a drift cell, ions migrate through a counter-lowing buffer gas in the presence of a low electric ield. The use of the drift cell in this manner is often referred to as ion mobility spectrometry (IMS), which is now a well-established analytical technique that is employed throughout the world for the detection of explosives, drugs, and

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chemical warfare agents. Ion mobility measures the time it takes for ions to traverse a drift tube. Ion separation occurs as a result of interactions between these ions and the buffer gas; the extent of separation depends not only on the mass and charge, as may be anticipated, but on the shape (or conformation) of the ion, which is unique to ion mobility spectrometry. The study of ion/molecule reactions in IMS is discussed in Chapter 13. Two alternative approaches have been introduced recently; these are high-ield asymmetric waveform ion mobility spectrometry (FAIMS) and traveling wave ion mobility spectrometry (TWIMS). In Volume IV, Chapter 5 was devoted to a discussion of FAIMS. In Chapter 8 is presented an account of traveling wave ion mobility spectrometry. Unlike drift cell ion mobility experiments, where a constant low electric ield is applied to the mobility cell, traveling wave ion mobility spectrometry uses a traveling wave comprising a series of transient direct current voltages to propel ions through a stacked-ring ion guide (SRIG) to which radiofrequency voltages have been applied to consecutive electrodes. The SRIG consists of a series of ring electrodes that are arranged orthogonally to the ion transmission axis, and opposite phases of radiofrequency voltage are applied to adjacent rings. When a transient direct current potential, superimposed upon this radiofrequency potential, is applied to one pair of adjacent ring electrodes, ions are propelled through the SRIG. The transient direct current potential moves along ring electrode pairs across the length of the SRIG at regular time intervals, generating a sequence of traveling waves (T-Waves). This particular coniguration of SRIG is referred to as a traveling wave ion guide (TWIG). A concatenation of three TWIGs has been incorporated within a Q-TOF geometry to create the Synapt™ HDMS system, a commercial instrument incorporating ion mobility separation. Most applications using the Synapt have focused on studying the conformation of proteins and protein complexes. Among the applications discussed here is a study of the prion protein, a ibril-forming protein involved in prion diseases. Prions are a class of fatal, infectious, neurodegenerative diseases that affect both humans and animals. Part 3. Ion Spectroscopy. In Chapter 9, we return to the theme of ion photodissociation, which was included also in Volume IV, Part 6, in an exploration of trapped-ion photodissociation, electron photodetachment, and luorescence. Trapped-ion luorescence may offer an alternative approach for the elucidation of ion conformation. Whereas these spectroscopic experiments require high ion densities, much attention is directed to the spectroscopic study of single ions conined in an ion trap. Chapters 10 and 11 are illustrative of such studies, with the former devoted to the study of a single molecular ion in a linear ion trap and the latter to a single atomic ion in Paul-type ion traps. While both types of studies require extensive cooling of the subject ion, once such cooling has been achieved, the ions can remain conined for many hours. Chapter 9 contains a discussion of practical aspects of experimental design for the pursuit of photodissociation, electron photodetachment, and luorescence of trapped, mass-selected organic ions. A review is given of the wide range of possible spectroscopic experiments that can be combined fruitfully with the ion storage and massselective capabilities provided by ion-trapping devices for the scrutiny of molecular ions. Details of the modiication of a quadrupole ion trap together with the results from extensive modeling of the apparatus are presented. Photodissociation is the

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fragmentation of an ion due normally to the absorption of light in a narrow wavelength range. In this chapter, the excitation source is stepped or scanned through a range of wavelengths, while monitoring ion intensities as a function of excitation wavelength, in order to construct an optical spectrum. When the extent of photodissociation is monitored as a function of excitation wavelength, the process is termed ‘action’ or ‘consequence’ spectroscopy. The application of action spectroscopy, which has been used to generate vibrational (infrared) and vibronic (ultraviolet-visible) spectra of mass spectrometric precursor and product ions, is discussed. Fluorescence spectroscopy, in which radiative emission from activated ions can be monitored using a photon detector, is shown here to be highly sensitive to a chromophore’s local environment, making it an excellent probe of ion conformation. In Chapter 10, the novel technique of sympathetically-cooled single ion mass spectrometry (SCSI-MS) is described; this technique relies on the measurement of the resonant excitation frequency of one of the two oscillatory modes of a trapped and crystallized linear two-ion system consisting of one laser-cooled atomic ion of known mass and the a priori unknown atomic or molecular ion, whose mass is to be determined. The mass of the unknown ion can be deduced from this measured frequency. The crystallization of the two-ion system results from the sympathetic cooling of the unknown ion through the Coulomb interaction with the laser-cooled ion; the two-ion system is aligned along the axis of the linear ion trap. Resonant excitation can be promoted by applying a sinusoidally-varying electric ield along this axis. The resonance frequencies are determined by monitoring luorescence from the laser-cooled ion while scanning the period of the applied driving force. When the period is equal to the period of one of the two oscillatory modes of the two-ion system, that is, the centerof-mass mode where the ions move in phase, or the breathing mode where the ions move with opposite phase, the motion of the ions becomes highly excited. Examples of molecular ions examined here are CaO+, MgD+, and MgH+. Chapter 11 gives a review of atomic clocks of the future using single ions conined in relatively small ion traps; small or miniature ion traps have been discussed in Volume IV, Chapter 2. The purpose of Chapter 11 is to expound upon the speciic topic of atomic clocks utilizing ion traps and the new challenges engaged presently for the measurement of time with extremely high precision. A major part of physics is dedicated permanently to the enhancement of measurement and more precise deinitions of the fundamental units. Among them, the time unit, the second, is one of the most crucial units necessary for the advancement of knowledge. The time unit was the irst for which the deinition put aside any material systems in that Greenwich Mean Time (GMT) was deined, in 1884, on the assumption that one second is equal to 1/86,400 of the mean solar day. As most of the fundamental constants can be related either to a time or to a frequency measurement, the quest for the detection of the smallest possible time variation in these constants, that is, the attainment of a time variation measurement of these constants at the 10 −17–10−18 level, is being continued. Highly accurate clocks are not merely a convenience; they are a necessity for such fundamental problems as the local position invariance, baseline interferometry, observation of the so-called ‘gravitational red-shift’, and for the ground-positioning system (GSP)-Galileo systems that require a panoply of atomic clocks located in satellites as well on the Earth’s surface. All of the current

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research into types of ion clocks and their development are covered explicitly in this chapter. Part 4. Practical Applications presents ive practical examples of trapped-ion technology that relect the wide diversity of applications of trapped-ion devices. Yet there is a common thread that links these applications, and it is the existence of such a thread that justiies the publication of the Volume IV and Volume V monographs. This common thread links the efforts, foresight, and business acumen of manufacturers with the knowledge and experimental skills of researchers to bring forth instruments at an affordable price that will enhance and protect the well-being of mankind. Such a claim is not an overstatement, as is shown by the inal three chapters, Chapters 14–16. In the Preface to the irst edition of Quadrupole Storage Mass Spectrometry,* a monograph that may be familiar to some of the more curious graduate students, we wrote “There is now abundant evidence of the application to the health services of mass spectrometric techniques with concomitant high sensitivity and resolution for toxicological studies; studies of metabolism and incipient disease; environmental problems; the quality of food, well water, and materials; forensic sciences; and so forth. Thus, the advent of the ion trap detector permits a much greater use of mass spectrometric techniques not only in the technically developed countries but also in those countries which are technically less advanced.” Chapter 12 affords an example of industry–university cooperation with a description of the commercially-available digital ion trap, which is the subject of Chapter 4 in Volume IV, employed for the fragmentation of mass-selected ions by boundary-activated dissociation, a technique that was discovered in an academic research laboratory. Chapter 13 gives an account of the utilization of ion mobility spectrometry and ion mobility/mass spectrometry for the study of basic ion/molecule reactions at ambient pressure, which is the pressure regime used commonly for the detection of explosives, drugs, and chemical warfare agents. Chapter 14 is concerned with a novel application, that of imaging mass spectrometry wherein thin tissue sections are analyzed directly and permit the creation of chemically-selective images of intrinsic chemical distributions. This technique allows characterization of known compounds from a variety of tissues, and the identiication of unknown chemical signatures for a variety of studies such as disease progress or pharmaceutical studies. Chapter 15 permits a review of the progress made in the instrumentation for gas chromatography/ion trap mass spectrometry since the introduction by Finnigan MAT of the irst commercial gas chromatograph/Ion Trap Detector™. Ion traps have found an important application as in situ chemical analyzers for a broad range of ields such as homeland security, industry, and environmental monitoring applications. Such devices can be used in marine science, where there is a high demand for monitoring natural compounds and the ever-increasing quantities of compounds of anthropogenic origin that enter rivers, lakes, and oceans. Chapter 16 presents a detailed account of the prolonged remote monitoring of volatile organic compounds in ield waters by membrane inlet mass spectrometry (MIMS) using a quadrupole ion trap.

∗ March R.E., Hughes R.J., Todd J.F.J., Quadrupole Storage Mass Spectrometry, 1989. New York, Wiley

Interscience.

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Chapter 12 describes the novel operation of a digital ion trap (DIT) for the determination of the boundaries of the stability diagram and for the utilization of boundary-activated dissociation. Ion motion in a Paul or quadrupole ion trap driven by a rectangular wave quadrupolar ield was described in the early 1970s, but it was not until 2000 that the mass-selective resonance method with the ion secular frequency under digital operation conditions was described. The circuits of the digital ion trap switch very rapidly between discrete direct current high voltage levels in order to generate the trapping waveform voltage applied to the ring electrode. An alternative ion activation method, boundary-activated dissociation, proposed in 1991, entails moving the working point (that is, the point (az, qz) on a quadrupole ion trap stability diagram deined by the magnitudes of the trapping parameters az and qz) of a mass-selected ion species close to one of the boundaries of the stability diagram. This method can be realized with the combined effect of suitable direct current and radiofrequency potentials applied to the ion trap electrodes. Under these conditions, dissociation of the mass-selected ion species can be induced. In order to evaluate the performance of the digital ion trap for boundary-activated dissociation experiments, the real shape of the stability diagram needed to be determined. As an ion species undergoes fragmentation when its working point is moved close to a stability boundary, this behavior was used to map the boundaries of the stability diagram for a digital ion trap. In the digital ion trap, variation of the duty cycle of the rectangular waveform readily allows the introduction of the direct current component for boundary-activated dissociation experiments. Regrettably, direct current power supplies are no longer made available in commercial ion trap instruments. Chapter 13 gives an introduction to the principles of ion mobility spectrometry, together with an overview of the type of information obtainable from ion mobility studies at atmospheric pressure and the variety of experimental methods employed in such studies. It is shown that thermodynamic data, which are obtainable from these studies and are suitable for tabulation, include standard enthalpies, entropies, and free energies; such data, when obtained at a speciied temperature, can be regarded as universally applicable when all participants are at thermal equilibrium. Thermal equilibrium is established readily in an ion mobility spectrometer at ambient pressure because each ion experiences more than 1010 collisions per second with neutral atoms or molecules of the supporting gas atmosphere. In addition, the residence time of an ion in an ion mobility spectrometer operating at atmospheric pressure is ca 5–50 ms, which allows the study of the interactions of ions with molecules at very low concentrations. Illustrated here is the further advantage of thermochemical determinations obtained by ion mobility spectrometry in that the available temperature range, from sub-ambient to more than 500 K, is far greater than that available with many other experimental methods. In the lower electrostatic ield conditions of ion mobility spectrometry, thermal conditions always prevail for ions. Hence, this technique permits ready determination of ion/molecule reaction rate constants, including those for clustering reactions, for both positive and negative ions, in the temperature range from below ambient to at least 600 K. Electron association and detachment reactions are studied more easily in an ambient pressure ion mobility spectrometer than the more conventional swarm-beam method.

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In Chapter 14 is described the principles and instrumental approaches of imaging mass spectrometry; it also provides real-world examples of the capabilities of quadrupole ion traps (QITs) and linear ion traps (LITs) for modern imaging mass spectrometry. Imaging mass spectrometry permits direct analysis of thin tissue sections from which chemically-selective images of intrinsic chemical distributions are created. Imaging mass spectrometry using matrix-assisted laser desorption/ionization (MALDI) provides the ability to characterize and to localize compounds within tissue sections, identifying potential but unknown markers of diseases such as cancer, or to determine where an administered drug (and its metabolites) has localized in a tissue section. An advantage that mass spectrometry provides to the ield of imaging is the identiication of non-targeted compounds. In a typical luorescence experiment, a luorophore must be administered that binds to a given compound for ready identiication of that speciic compound. With imaging mass spectrometry, the targeted compound can be localized and other compounds that may localize with the targeted compound can be identiied, thus providing a more complete understanding of the chemical signature of the speciic state under investigation. Examples are discussed of the remarkable utility of sequential tandem mass spectrometry to effect MSn for the identiication and structural characterizations of compounds from intact tissues. In addition, MSn is invaluable for the identiication of isobaric ions: for example, m/z 828 in one sample was shown to consist of four isobaric ions and each was identiied using MS3. Chapter 15 is devoted to a review of the development of the quadrupole ion trap as a detector for compounds eluting from a gas chromatograph, together with an account of the progress made by Varian Inc. in ion trap technology for gas chromatography/ mass spectrometry and gas chromatography/tandem mass spectrometry. Described here is the new type of non-linear ion trap, that is, the ield is made non-linear by the superimposition of a dipole and higher-order multipoles upon the quadrupole ield by a switchable electric circuit. A detailed discussion of ion traps with electrically-induced non-linear ields is given in Volume IV, Chapter 14. In the ion trap, both dipole and quadrupole supplemental ields are applied to the two end-cap electrodes with their frequencies tuned to βz = 2/3; the resonance of each ion species in turn with both the dipole and quadrupole supplemental ields results in improved mass resolution, higher scan speed, and extended charge capacity. Electron impact ionization and chemical ionization, within the ion trap and external to it, are discussed. The chapter is replete with many examples of applications and contains 47 igures. Chapter 16 describes the monitoring of some 16 volatile organic compounds that are included in the European Union Directive 98/83 and classiied as being potentially deleterious to human health when present in drinking water. The technique employed was that of membrane inlet mass spectrometry (MIMS) combined with a quadrupole ion trap. While this technique is well known for its simplicity and sensitivity, no previous account has been published of the implementation of this technique to work unattended for months. Four instruments were deployed in unmanned sites, where they monitored volatile organic compounds (VOCs) in natural waters and wastewater during a period exceeding 1 year for each instrument. The instruments were equipped with software that facilitated the automatic operation of each analysis, the identiication and quantitation of VOCs from the raw mass spectra, and the transmission of the results to a remote control room via an Internet connection. In the remote control

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room, a personal computer displayed the results as bar graphs and was programed to activate alarms when set concentration thresholds were exceeded. The chapter discusses laboratory performance and ield performance: the former in terms of sensitivity, reproducibility, linearity tests, and comparison with purge-and-trap combined with gas chromatography/mass spectrometry; and the latter in terms of data output, most frequent maintenance operations and technical failures, and overall stability of the four remotely-controlled instruments. The longest period of unattended remote monitoring was of 526 days, of an industrial wastewater treatment plant. We wish to thank the many people who have assisted us in one way or another with myriad tasks that must be carried out in order to arrive at the publication of a monograph from a collection of manuscripts in a variety of formats and styles. First of all, to our contributors, without whom this monograph would not have appeared in print. We give thanks for their individual inspiration; we thank them for the fruits of their labors, and for their patient toleration of the idiosyncrasies of our editing, often involving repeated iterations between the two of us and the authors themselves. The 16 chapters that constitute Volume V have originated from 36 authors and co-authors; a total of 91 authors and co-authors contributed to Volumes IV and V. For many of these co-authors this project has been a novel experience, thus we thank our lead authors for responding to our urging that they collaborate with young scientists in their laboratories. From where else will the monographs of tomorrow originate? At CRC Press, we thank Fiona Macdonald, Pat Roberson, Rachael Panthier, Lindsey Hofmeister, Hilary Rowe, and Jennifer Derima; at Datapage, we thank Ramkumar Soundararajan, the Project Manager. Finally, we express our sincere appreciation for the tolerance of our respective spouses, Kathleen March and Mavis Todd, and for their patience, support, and sacriices while this project, known informally as ‘PRATIMS’, took over our lives. Raymond E. March John F.J. Todd

Volume IV Contents Practical Aspects of Trapped Ion Mass Spectrometry Volume IV: Theory and Instrumentation Edited by Raymond E. March and John F.J. Todd Table of Contents PART I

Fundamentals

Chapter 1

An Appreciation and Historical Survey of Mass Spectrometry Raymond E. March and John F.J. Todd

Chapter 2

Ion Traps for Miniature, Multiplexed and Soft Landing Technologies Scott A. Smith, Chris C. Mulligan, Qingyu Song, Robert J. Noll, R. Graham Cooks, and Zheng Ouyang

PART II New Ion Trapping Techniques Chapter 3

Theory and Practice of the Orbitrap™ Mass Analyzer Alexander Makarov

Chapter 4

Rectangular Waveform Driven Digital Ion Trap (DIT) Mass Spectrometer: Theory and Applications Francesco Brancia and Li Ding

Chapter 5

High-Field Asymmetric Waveform Ion Mobility Spectrometry Randall W. Purves

Chapter 6

Ion Traps with Circular Geometries Daniel E. Austin and Stephen A. Lammert xxi

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PART III Fourier Transform Mass Spectrometry Chapter 7

Ion Accumulation Approaches for Increasing Sensitivity and Dynamic Range in the Analysis of Complex Samples Mikhail E. Belov, Yehia M. Ibrahim, and Richard D. Smith

Chapter 8

Radio Frequency-Only-Mode Event and Trap Compensation in Penning Fourier Transform Mass Spectrometry Adam M. Brustkern, Don L. Rempel, and Michael L. Gross

Chapter 9

A Fourier Transform Operating Mode Applied to a ThreeDimensional Quadrupole Ion Trap Y. Zerega, J. Andre, M. Carette, A. Janulyte, and C. Reynard

PART IV

Quadrupole Rod Sets

Chapter 10 Trapping and Processing Ions in Radio Frequency Ion Guides Bruce A. Thomson, Igor V. Chernushevich, and Alexandre V. Loboda Chapter 11 Linear Ion Trap Mass Spectrometry with Mass-Selective Axial Ejection James W. Hager Chapter 12 Axially-Resonant Excitation Linear Ion Trap (AREX LIT) Yuichiro Hashimoto

PART V

3D-Quadrupole Ion Trap Mass Spectrometry

Chapter 13 An Examination of the Physics of the High-Capacity Trap (HCT) Desmond A. Kaplan, Ralf Hartmer, Andreas Brekenfeld, Jochen Franzen, and Michael Schubert Chapter 14 Electrically-Induced Nonlinear Ion Traps Gregory J. Wells and August A. Specht

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Chapter 15 Fragmentation Techniques for Protein Ions Using Various Types of Ion Trap J. Franzen and K. P. Wanczek Chapter 16 Unraveling the Structural Details of the Glycoproteome by Ion Trap Mass Spectrometry Vernon Reinhold, David J. Ashline, and Hailong Zhang Chapter 17 Collisional Cooling in the Quadrupole Ion Trap Mass Spectrometer (QITMS) Philip M. Remes and Gary L. Glish Chapter 18 ‘Pressure Tailoring’ for Improved Ion Trap Performance Dodge L. Baluya and Richard A. Yost Chapter 19 A Quadrupole Ion Trap/Time-of-Flight Mass Spectrometer Combined with a Vacuum Matrix-Assisted Laser Desorption Ionization Source Dimitris Papanastasiou, Omar Belgacem, Helen Montgomery, Mikhail Sudakov, and Emmanuel Raptakis

PART VI

Photochemistry of Trapped Ions

Chapter 20 Photodissociation in Ion Traps Jennifer S. Brodbelt Chapter 21 Photochemical Studies of Metal Dication Complexes in an Ion Trap Guohua Wu, Hamish Stewart, and Anthony J. Stace

Editors Raymond E. March, PhD, DSc, D(hc), FCIC, is presently Professor Emeritus of Chemistry at Trent University in Peterborough, ON, Canada. He obtained a BSc (Hons) in Chemistry from Leeds University in 1957; a PhD from the University of Toronto in 1961 (supervised by Professor John C. Polanyi, Nobelist 1986); a DSc from Leeds University in 2000; and an honorary doctorate (D(hc)) from l’Université de Provence in 2008. From 1954 to 1957, he was a Cadet Pilot in the Leeds University Air Squadron Royal Air Force Volunteer Reserve (RAFVR) and, from 1958 to 1963, a Flight Lieutenant in the Royal Canadian Air Force (Auxiliary) (RCAF). From 1960 to 1961, he held a Canadian Industries Limited Research Fellowship. From 1962 to 1963, he was a Post-Doctoral Fellow with Professor H.I. Schiff at McGill University, and a Research Associate from 1963 to 1965, during which time he lectured at McGill University and Loyola College. In 1965, he joined the faculty of Trent University where he has conducted independent research for some 44 years in gas-phase kinetics, optical spectroscopy, gaseous ion kinetics, analytical chemistry, nuclear magnetic resonance spectroscopy, and mass spectrometry. Kathleen and Ray have been married for 51 years; they have three daughters, Jacqueline, Roberta, and Sally with spouses Paul, Stuart, and Lauren, respectively, and nine grandchildren, Shawn, Jessica, Thomas, Daniel, Rebecca, Sara, James, Madeline, and Carson, in order of appearance. Dr. March has published and/or co-authored over 170 scientiic papers and some 75 conference presentations in the above areas of research with emphasis on mass spectrometry, both with sector instruments and quadrupole ion traps. Dr. March is a co-author with Dr. Richard J. Hughes and Dr. John F.J. Todd of Quadrupole Storage Mass Spectrometry, published in 1989. A second edition of Quadrupole Storage Mass Spectrometry, co-authored by Dr. March and Dr. John F.J. Todd was published in 2005. Dr. March and Dr. John F.J. Todd co-edited three volumes entitled Practical Aspects of Ion Trap Mass Spectrometry, published in 1995. Volume IV in the series Practical Aspects of Trapped Ion Mass Spectrometry is in press. Dr. March is a co-author with Oscar V. Bustillos and André Sassine of A Espectrometria de Massas Quadupolar, published in Portuguese in 2005. Professor March is a Fellow of the Chemical Institute of Canada and a member of the American, British, and Canadian Societies for Mass Spectrometry. In 2009, he received the Gerhard Herzberg Award of the Canadian Society for Analytical Sciences and Spectroscopy (CSASS). In

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1995, he received the Distinguished Faculty Research Award from Trent University, and the Canadian Mass Spectrometry Society presented him with the Recognition Award and, in 1997, with the Distinguished Contribution Award. Dr. March is a member of the Editorial Advisory Boards for Rapid Communications in Mass Spectrometry, the Journal of Mass Spectrometry, and the International Journal of Mass Spectrometry. In 1975, Dr. March was an Exchange Fellow (NRC-CNRS) at Orsay, France, with Professor Jean Durup; in 1983, an Exchange Fellow (NRCRoyal Society of London) in Swansea, Wales, with Professor J.H. Beynon; in 1989 and 1992, a Visiting Professor, Université de Provence, Marseille, France, with Prof Fernande Vedel; in 1993 and 1995, a CNRS Visiting Professor, Université Pierre et Marie Curie, Paris, France, with Prof Jean-Claude Tabet; and in 1999, a Visiting Professor, Université de Provence, Marseille, France, with Yves Zerega. In 1987, Dr. March was a Distinguished Lecturer at the Universities of Berne, Neuchatel, and Lausanne, in Switzerland. Dr. March’s research in the ield of mass spectrometry and gas-phase ion chemistry involved the development and application of mass spectrometric instruments, particularly quadrupole ion trap mass spectrometers and hybrid mass spectrometers, for both fundamental studies and the formulation of analytical protocols for the determination of compounds of environmental interest. His current research interests are focused within Trent University’s Water Quality Centre (www. trentu.ca/wqc/). As a founding member of the Water Quality Centre his principal research interest lies in the mass spectrometric and nuclear magnetic resonance spectroscopic investigation of natural compounds that, having been formed by plants, may enter waterways and/or the water table. His current research involves the study of lavonoids and lavonoid glycosides; such compounds are often found in those products that have become known as neutraceuticals. Electrospray ionization combined with tandem mass spectrometry permits the investigation of ion fragmentation at high mass resolution and the derivation of possible ion fragmentation mechanisms using ion structures; these studies are supported by theoretical calculations carried out in collaboration with Professor E.G. Lewars. An important aspect of this research is the development of appropriate analytical protocols for lavonoid glycosides in water and in plant extracts. Nuclear magnetic resonance (NMR) studies of lavonoids and metabolites, carried out in collaboration with Professor D.A. Ellis and Dr. D.C. Burns, have permitted a rationalization of chemical shifts with product ion mass spectra and the development of a predictive model for 13C chemical shifts in lavonoids. At present, Dr. March is carrying out an investigation of volatile compounds formed by Ash trees in response to an attack by the Emerald Ash Borer. These researches are supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grants Program), the Canada Foundation for Innovation, the Ontario Research and Development Challenge Fund, Ontario Ministry of Natural Resources, and Trent University. Dr. March has enjoyed longterm collaborations with Professor John Todd, with colleagues at l’Université de Provence and l’Université Pierre et Marie Curie (France), and with colleagues in Padova (Italy).

Editors

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John F.J. Todd, BSc, PhD, CChem, FRSC, CEng, FInstMC, is currently Emeritus Professor of Mass Spectroscopy at the University of Kent, Canterbury, U.K. He obtained his Class I Honours BSc degree in Chemistry in 1959 from the University of Leeds, from whence he also gained his PhD degree and was awarded the J.B. Cohen Prize in 1963; he was a member of the radiation chemistry group led by the late Professor F.S. (later Lord) Dainton, FRS. From 1963 to 1965, he was a Fulbright Research Scholar and Post-Doctoral Research Fellow in Chemistry with the late Professor Richard Wolfgang at Yale University. In 1965, he was one of the irst faculty members appointed to the then new University of Kent at Canterbury, U.K. John and Mavis Todd have been married for 46 years and have three sons: John (Andrew), Eric, and Richard, two daughters-in-law Dorota and Marie, and six grandchildren, Alice, Max, Maja, Luke, Daniel, and Lara. Professor Todd’s research interests, spanning some 44 years, have encompassed positive and negative ion mass spectral fragmentation studies, gas discharge chemistry, ion mobility spectroscopy, analytical chemistry, and ion trap mass spectrometry. His work on 3D quadrupole (Paul) ion traps commenced in 1968, when he irst developed the “Quistor/Quadrupole” instrument for the characterization of the behavior of ions conined in radiofrequency electric ields and as a vehicle for the study of gasphase ion chemistry. As a consultant to Finnigan MAT during the 1980s and 1990s, he was a member of the original team that developed the irst commercial ion trap mass spectrometer. In another consultancy role, Professor Todd is involved currently with one of the most extended single mass spectrometric investigations ever undertaken: the use of an ion trap mass spectrometer for the isotope ratio measurement of cometary material as part of the “Rosetta” project (launched 2004, scheduled arrival at its target comet in 2014). Professor Todd has published and/or co-authored some 116 scientiic papers and over 118 conference contributions, concentrating mainly on various aspects of mass spectrometry. With Professor Dennis Price, he co-edited four volumes of Dynamic Mass Spectrometry and he edited Advances in Mass Spectrometry 1985 (which contained the proceedings of the 10th International Mass Spectrometry Conference, Swansea, at which he was also a plenary lecturer). In addition, he was an editor of the International Journal of Mass Spectrometry and Ion Processes from 1985 to 1998, has served on the Editorial Boards of Organic Mass Spectrometry/Journal of Mass Spectrometry and Rapid Communications in Mass Spectrometry, and is currently a member of the Board for the European Journal of Mass Spectrometry. With Dr. Raymond E. March, Dr. Todd co-edited three volumes entitled Practical Aspects of Ion Trap Mass Spectrometry, published by CRC Press in 1995. In addition, Dr. Todd was a co-author with Dr. Raymond E. March and Dr. Richard J. Hughes of Quadrupole Storage Mass Spectrometry, published by Wiley in 1989; a second edition of Quadrupole Storage Mass Spectrometry, co-authored by Dr. Todd and Dr. Raymond E. March was published in 2005. Volume IV in the series Practical Aspects of Trapped Ion Mass Spectrometry is in press.

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Professor Todd is a Chartered Chemist and a Chartered Engineer, and has served terms as Chairman and as Treasurer of the British Mass Spectrometry Society. In 1988, he was a Canadian Industries Limited Distinguished Visiting Lecturer at Trent University, Peterborough, Ontario. In 1997, Dr. Todd was awarded the Thomson Gold Medal by the International Mass Spectrometry Society for “outstanding contributions to mass spectrometry,” and in 2006 he was awarded the Aston Medal by the British Mass Spectrometry Society, of which he is also a Life Member. In 2008, he was accorded Honorary Life Membership of the Royal Society of Chemistry. Outside the immediate conines of his academic work, Professor Todd was appointed as Master of Rutherford College, University of Kent (1975–1985), and as the irst Chairman of the newly created Canterbury and Thanet Health Authority (UK National Health Service) between 1982 and 1986. During the period 1995–2006 he was the founding Chairman of the newly established Board of Governors of St Edmund’s School Canterbury, and until August 2007 he was a Governor of Canterbury Christ Church University; he was admitted as an Honorary Fellow of Canterbury Christ Church University in 2008. From 1979 to 1989, Professor Todd was Chairman of the Mass Spectrometry Sub-Committee, Commission I.5 of the International Union of Pure and Applied Chemistry (IUPAC), and between 1995 and 2007 he was Chairman of the Management Advisory Panel for the EPSRC National Mass Spectrometry Service Centre, based at the University of Wales Swansea. He has enjoyed long-term collaborations with co-editor Professor Raymond March, with colleagues at Finnigan MAT in the United Kingdom and the United States, and with groups in Nice (France) and Padova and Torino (Italy).

Contributors Alberto Berton CNR-ISTM Corso Stati Uniti 4 Padova, Italy

Michael Drewsen Department of Physics and Astronomy University of Aarhus Aarhus, Denmark

Paola Bocchini Department of Chemistry ‘G. Ciamician’ University of Bologna Bologna, Italy

Gary A. Eiceman Department of Chemistry and Biochemistry New Mexico State University Las Cruces, New Mexico

Francesco L. Brancia Shimadzu Research Laboratory (Europe) Manchester, United Kingdom

Matthew W. Forbes Department of Chemistry University of Toronto Toronto, Ontario, Canada

Jennifer S. Brodbelt Department of Chemistry and Biochemistry University of Texas at Austin Austin, Texas Joseph E. Chipuk Department of Chemistry and Biochemistry University of Texas at Austin Austin, Texas Joshua J. Coon Department of Chemistry and Biomolecular Chemistry University of Wisconsin Madison, Wisconsin Helen J. Cooper School of Biosciences University of Birmingham Edgbaston, Birmingham, United Kingdom

Jennifer M. Froelich Department of Chemistry Michigan State University East Lansing, Michigan Guido C. Galletti Department of Chemistry ‘G. Ciamician’ University of Bologna Bologna, Italy Timothy J. Garrett Department of Medicine University of Florida Gainesville, Florida John E. George III Varian Inc., Scientiic Instruments Walnut Creek, California Klaus Højbjerre Department of Physics and Astronomy University of Aarhus Aarhus, Denmark xxix

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Contributors

Rebecca A. Jockusch Department of Chemistry University of Toronto Toronto, Ontario, Canada

Luca Raveane CNR-ISTM Corso Stati Uniti 4 Padova, Italy

Jian Liu Department of Chemistry Purdue University West Lafayette, Indiana

Gavin E. Reid Department of Chemistry, Biochemistry and Molecular Biology Michigan State University East Lansing, Michigan

Yali Lu Department of Chemistry Michigan State University East Lansing, Michigan Graeme C. McAlister Department of Chemistry and Biomolecular Chemistry University of Wisconsin Madison, Wisconsin Scott A. McLuckey Department of Chemistry Purdue University West Lafayette, Indiana Joel H. Parks The Rowland Institute at Harvard Cambridge, Massachusetts Francesca Pinelli Department of Chemistry ‘G. Ciamician’ University of Bologna Bologna, Italy Benoît Plet European Institute of Biology and Chemistry University of Bordeaux Pessac, France Romina Pozzi Department of Chemistry ‘G. Ciamician’ University of Bologna Bologna, Italy

Jean-Marie Schmitter European Institute of Biology and Chemistry University of Bordeaux Pessac, France James H. Scrivens Department of Biological Sciences University of Warwick Coventry, United Kingdom Peter Frøhlich Staanum Department of Physics and Astronomy University of Aarhus Aarhus, Denmark John A. Stone Department of Chemistry Queens University Kingston, Ontario, Canada Francis O. Talbot Department of Chemistry University of Toronto Toronto, Ontario, Canada Konstantinos Thalassinos Department of Biological Sciences University of Warwick Coventry, United Kingdom

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Pietro Traldi CNR-ISTM Corso Stati Uniti 4 Padova, Italy Fernande Vedel Physique des interactions ioniques et moléculaires (PIIM) Université de Provence Marseille, France

Mingda Wang Varian Inc., Scientiic Instruments Walnut Creek, California Richard A. Yost Department of Chemistry University of Florida Gainesville, Florida

Part I Ion Reactions

Reactions in 1 Ion/Ion Electrodynamic Ion Traps Jian Liu and Scott A. McLuckey CONTENTS 1.1 1.2

Introduction ........................................................................................................3 Tools for the Study of Ion/Ion Reactions ...........................................................4 1.2.1 Ion/Ion Reactions in Three-Dimensional (3D) Quadrupole Ion Traps.............................................................................5 1.2.2 Ion/Ion Reactions in Linear Ion Traps (LITs) ........................................9 1.2.3 Ion/Ion Reactions in Hybrid Instruments ............................................13 1.3 Methodologies/Applications .............................................................................15 1.3.1 Charge State Manipulation: Proton Transfer .......................................15 1.3.1.1 Macromolecule Mixture Analysis.........................................15 1.3.1.2 Precursor Ion Charge State Manipulation.............................16 1.3.1.3 Simpliication of Product Ion Mass Spectra .........................17 1.3.2 Charge Inversion ..................................................................................19 1.3.3 Metal–Ion Transfer ...............................................................................19 1.3.4 Electron Transfer Dissociation (ETD) .................................................21 1.4 Conclusions...................................................................................................... 24 References ..................................................................................................................25

1.1 INTRODUCTION Interactions between gas-phase ions of opposite polarities occur commonly in various environments such as the atmosphere, plasmas, lames [1–4], etc. It has been more than a century since the irst study of the interaction between oppositely-charged ions, which can be dated back to the work by Thomson and Rutherford [5]. However, the study of ion/ion reactions can be challenging particularly when the reactions take place between singly-charged cations and anions, as was the case in the majority of the early studies, because the products are neutral species and, therefore, dificult to analyze and detect. With the advent of electrospray ionization (ESI) [6–9] and its propensity for producing multiply-charged ions from high mass molecules, attention has been directed to the reactions between oppositely-charged ions involving multiply-charged ions, which produce charged products readily amenable to study by mass spectrometry. Consequently, a rapidly-growing range of reaction phenomena are being observed in the ion/ion reactions of multiply-charged ions, which is permitting new insights to be drawn regarding ion/ion reaction thermodynamics and dynamics. While ion/ion 3

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

reactions involving large multiply-charged ions are not important in plasmas or lames, for example, they are enabling an expanding array of new analytical applications, particularly in bioanalysis. Although ion/ion reactions can be implemented readily under various conditions, including near atmospheric pressure [10–15], ion/ion reactions performed in electrodynamic ion traps afford the opportunity for selective ion manipulation due to the mass/charge-dependent frequencies of motions executed by stored ions. Therefore, ion traps allow for the study of ion/ion reactions within the context of tandem mass spectrometry (MSn) experiments. As a result, many ion/ion reaction studies have been undertaken in electrodynamic ion trap-based instruments. Several reviews [16–18] of ion/ion reactions involving multiply-charged ions have been published, that focus on aspects of instrumentation, applications, reaction phenomena, and fundamentals including thermodynamics and kinetics. Rather than providing a comprehensive discussion of all aspects related to ion/ion chemistry, this chapter aims primarily to provide a brief description of the instrumentations, methodologies, and applications of ion/ion reactions in ion traps, especially within the context of biomolecule analysis, with particular emphasis on developments since the publication of recent reviews.

1.2 TOOLS FOR THE STUDY OF ION/ION REACTIONS Fundamental requirements for any tool intended for ion/ion reaction studies are the ability to generate ions of opposite polarities within a single experiment, and to furnish an interaction environment delivering good spatial and temporal overlap for the oppositely-charged ion populations. The environment for the ion/ion interaction can be created either outside or inside a mass spectrometer. The irst ion/ion reactions involving multiply-charged ions, for example, were demonstrated at near atmospheric pressure (ca 2 Torr) using a Y-tube low reactor [10,11], which admit, into separate inlet arms of the reactor, ions of opposite polarities produced by two ion sources, for example, ESI, and discharge sources. The ion/ion reactions took place once the two ion streams merged in the outlet arm of the reactor, which was coupled to the interface of the mass spectrometer, before sampling into the instrument. Implementation of ion/ion reactions external to the mass spectrometer separates physically the ionization process and ion/ion reactions from the mass analysis step. Advantages derived from this separation include the simplicity with which such ion/ion reactors can be adapted to any mass spectrometer coupled with ESI, independent optimization of mass analysis, and virtually no limits are imposed by the characteristics of the mass analyzer on the kinds of ions that can be used as reactants. However, reaction conditions can be dificult to deine in reactors operating at near atmospheric pressure due to the existence of a complicated reaction environment, where a mixture of ions, solvent vapors, and atmospheric gases are present in the reaction region. This situation can lead to ambiguities in the determination of mechanisms that give rise to products in some cases. Moreover, implementing ion/ ion reactions outside a mass spectrometer does not allow for a true tandem mass spectrometric experiment to be performed involving an ion/ion reaction between mass analysis stages. Many of the drawbacks associated with the implementation of

Ion/Ion Reactions in Electrodynamic Ion Traps

5

ion/ion reactions outside a mass spectrometer can be overcome by using an electrodynamic ion trap as a reaction vessel, at the cost of somewhat greater experimental complexity.

1.2.1 ION/ION REACTIONS IN THREE-DIMENSIONAL (3D) QUADRUPOLE ION TRAPS The majority of the early studies of ion/ion reactions under low pressure (ca 1 mTorr) conditions were performed with three-dimensional (3D) ion traps (that is, conventional Paul traps), which store ions in three dimensions by a radio-frequency (RF) voltage applied to a central ring electrode sandwiched between two end-cap electrodes. The 3D ion trap is inherently compatible with the study of ion/ion reactions due to its unique ability to store simultaneously ions of both polarities in overlapping regions of space [19,20]. The trapped ion assumes a characteristic set of m/z-dependent frequencies of motions in the oscillating quadrupole ield of the ion trap, which allows ready manipulation of ions of speciic massto-charge ratios for ion isolation and activation, both of which are common elements in a tandem mass spectrometric experiment. The ‘tandem-in-time’ nature of the ion trap MSn experiment [21,22] provides well-deined conditions for ion/ion reactions and is particularly useful in the determination of ion genealogy. Furthermore, the use of a bath gas, such as helium at ca 1 mTorr in the ion trap, intended originally to cool the ions translationally into the center of the trap to improve the mass resolution for the mass analysis [23], also improves ion/ion reaction eficiencies by maximizing the spatial overlap and minimizing the translational energies of the two ion clouds [24]. While the ion trap is particularly well-suited to serve as a reaction vessel for ion/ion reactions, it places constraints on the range of reactions that can be studied. For example, all of the reactant and product ions must fall within the limited range of m/z-values that can be stored simultaneously in an ion trap. The normal operation of the ion trap places a lower limit to the mass-to-charge value for ion storage, also known as low mass cut-off (LMCO) [25] of the ion trap, which is deined sharply by the operating RF (that is, RF frequency and amplitude) and ion trap dimensions. Any ion having an m/z-value less than the LMCO assumes an unstable trajectory and will be ejected from the ion trap. In the absence of a DC ield, all ions having m/z-values greater than the LMCO lie within the region of ion stability of the ion trap. However, ions of different mass-to-charge values experience different trapping potentials in the ion trap, as approximated by the so-called pseudo-potential trapping well ( Dz ) [25], which is deined also by the amplitude and frequency of the RF operating voltage, and the ion trap dimensions. The magnitude of the pseudo-potential trapping well is approximately inversely proportional to the mass-tocharge ratio at qz < 0.4. When the kinetic energy of an ion is close to or exceeds the magnitude of ( Dz ), it cannot be stored eficiently. Therefore, the shallow pseudo-potential trapping well associated with ions of high mass-to-charge ratios sets a practical upper limit to the range of ions that can be stored in the ion trap. Note, however, that a trapping mechanism for ions of high mass-to-charge ratio, in addition to that provided by the oscillating quadrupolar ield, can be created from the electrostatic ield created by ions of lower m/z-value stored in the ion trap. This mechanism has been referred to as ‘trapping by proxy’ [26] and can be important when the magnitude of Dz for the ions of high m/z-value is too low for eficient ion storage. Ions of low m/z-value are stored in

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deeper pseudo-potential wells than the ions of high m/z-value, and, when the density of ions of low m/z-value is suficiently high, oppositely-charged ions of high m/z-value can be prevented from escaping the ion trap. As a result, the upper limit of the m/z-value for mutual ion storage can be increased when a high-density of ions of low m/z-value of opposite polarity is available. For a related reason, the simultaneous presence of ions of each polarity can affect mass analysis, particularly for the ions of lower charge densities. The electric ield of the higher density ion population can affect signiicantly the motions of the lower density ion population such that they do not oscillate at the frequencies expected based solely on the presence of an oscillating quadrupolar electric ield [27]. This complication makes it necessary to eject the ion population of higher density prior to mass analysis of the ion population of lower density when the ion trap serves as both a reaction vessel and a mass analyzer. Despite these constraints, the advantages associated with physical separation of the ionization process from the ion/ion reaction region and ready manipulation of ions of speciic m/z-values by use of an ion trap instrument make it a powerful tool in the study of ion/ion reactions. The 3D ion trap can be used directly without any modiication as a reactor for ion/ ion reactions, but accommodations must be made to facilitate the admission and transmission of ions of each polarity into the ion trap. Ion admission into the ion trap is accomplished usually in one of these two ways: (i) introducing ions of one polarity through a hole in an end-cap electrode and admitting ions of opposite polarity into the trap through the aperture in the ring electrode; or (ii) directing ions of each polarity sequentially through a hole in one of the end-cap electrodes with the guidance of a ‘turning’ quadrupole. By use of either ion introduction or transmission approach, a variety of ion sources have been adapted to the 3D ion trap to generate ions of different types (such as, positive vs negative; open-shell vs closed-shell) for various applications. For example, shown schematically in Figure 1.1 is one of the most commonly-used designs [28] for the study of ion/ion reactions between multiply-charged cations and singly-charged anions. This instrument incorporates an ESI source to produce multiply-charged cations, which were introduced into the ion trap through the end-cap electrode adjacent to the source, and an atmospheric sampling glow discharge ionization (ASGDI) source [29] to generate singly-charged negative ions, which entered the ion trap through an aperture in the ring electrode. A similar coniguration was adopted in a modiied Hitachi M-8000 ion trap mass spectrometer [30] (San Jose, CA, USA) with improved igures-of-merit for mass analysis. A typical experimental sequence with this instrumental setup involved cation injection, ion isolation, anion injection, mutual storage of both polarities, and mass analysis through resonance ejection [31]. Particular emphasis was directed to proton transfer reactions for charge-state manipulation of multiply-protonated protein/peptide ions, both precursor and product ions, using proton transfer reagents such as anions derived from perluoro-1,3-dimethylcyclohexane (PDCH). Recently, a setup similar to that shown in Figure 1.1 has been particularly useful in the study of another important reaction phenomenon, electron transfer dissociation (ETD) [32,33], by reacting multiply-protonated polypeptides with radical anions produced by the ASGDI source from species such as sulfur dioxide and nitrobenzene [33,34]. An apparatus also similar to that shown in Figure 1.1 has been developed by Glish and co-workers [35] to study the ion/ion reactions of iron and iron-containing ions with oppositely-charged peptide and protein ions, in which the multiply-charged

7

Ion/Ion Reactions in Electrodynamic Ion Traps Ion trap analyzer Guard ring Electrospray needle

Electron multiplier

Protein sample infusion

Conversion dynode Gate lens

Application of high voltage DC pulser (gate lens)

Positive ions Negative ions Needle valve Inlet for air/PDCH vapor

PDCH/air vapor

FIGURE 1.1 Schematic of a quadrupole ion trap mass spectrometer for ion/ion reactions between multiply-charged cations generated by ESI (ion injection through the ion entry endcap electrode) and singly-charged anions produced by ASGDI (ion injection through the ring electrode). (Reproduced from Stephenson J.L.; McLuckey, S.A., Int. J. Mass Spectrom. Ion Processes. 1997, 162, 89–106. With permission from Elsevier.)

cations were produced via ESI and introduced into the ion trap through the hole in an end-cap electrode while the singly-charged anions were formed using laser desorption from a stainless steel surface and admitted into the ion trap via the aperture in the ring electrode. In addition to reagent ions, electrons from a gated ilament have been introduced also into the 3D ion trap [36] through a hole in the ring electrode for an in situ formation of singly-charged positive ions [37], as demonstrated in the irst study of ion/ion reactions using a 3D ion trap. Despite its utility in the study of multiplycharged anions reacting with singly-charged cations, this approach is not well-suited to the study of ion/ion reactions of multiply-charged cations due to the ineficient nature of in situ anion formation with electrons from a heated ilament [38]. Signiicant progress has been made in the exploration of gas-phase ion/ion chemistry with the development of the apparatus described above; however, ion injection via the ring electrode [36] in these approaches places constraints on the range of species available for study due to the existence of strong electric ields near the ring electrode, which lead to both low eficiency in ion transmission (approximately two orders of magnitude lower than axial injection through the end-cap electrode) and a high propensity for ion fragmentation [30]. The constraints imposed by the use of the ring electrode for ion introduction can be removed by admitting ions of both polarities through an end-cap electrode with the use of a DC ‘turning’ quadrupole [39], which directs the ions along a common ion path into the ion trap. As one example of

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

such an implementation, ions of opposite polarity were produced from two different ion sources that were both perpendicular to the axial dimension of the ion trap and 180º from each other [39]. The employment of a ‘turning’ quadrupole allows for lexible combinations of up to three ion sources, including combinations like ESI/ESI/ atmospheric pressure chemical ionization (APCI), ESI/ASGDI, ESI/corona discharge ionization, and ESI/ESI, the latter of which allowed for the irst study of ion/ion reactions between multiply-charged positive and negative ions [39,40]. As the scope of ion/ion reaction studies expands, it is sometimes desirable that product ions from an ion/ion reaction be ‘processed’ further by various other reagent ions produced from different ion sources (that is, sequential ion/ion reactions involving different reagent ions). A simple example of such an application is charge reduction of a protein complex formed by gas-phase ion/ion reactions, which requires two ESI sources for multiply-charged positive and negative protein ion formation and another ion source (for example, ASGDI source) to generate the charge-reducing reagent [41]. Such experiments can be accomplished readily in a 3D ion trap by taking advantage of its MSn capability, provided enough ion sources can be coupled to the instrument and ions of different types can be delivered sequentially to the ion trap in a timely manner. Such an apparatus has been developed [41], as shown in Figure 1.2, with up to four independent ion sources, one of which is an ASGDI source capable of delivering ions through the ring electrode and the other three can be any combination of ESI, ASGDI, and corona discharge ionization sources, from which ions are introduced sequentially into the ion trap via the end-cap electrode guided by a DC turning quadrupole. As the analytical utility of ion/ion reactions has become increasingly apparent, 3D ion traps intended for ion/ion reaction applications, such as ETD, have become commercially available, such as the Bruker HCTultra™/Agilent 6340 ETD ion trap.

Glow discharge source

Turning quad

–ESI source

ESI/discharge source

Ion trap

Tube lens + ESI source

FIGURE 1.2 Schematic diagram of multiple-source quadrupole ion trap mass spectrometer. (Reproduced from Badman, E.R.; Chrisman, P.A.; McLuckey, S.A., Anal. Chem. 2002, 74, 6237–6243. With permission from American Chemical Society.)

Ion/Ion Reactions in Electrodynamic Ion Traps

9

This ion trap instrument has a chemical ionization source located orthogonally to an octopole ion guide, which admits luoranthene anions formed in the chemical ionization source and transmits them to the entrance of the end-cap electrode [42]. The positive polypeptide ions produced from ESI are admitted axially to the ion trap through the octopole ion guide and the entrance of the end-cap electrode.

1.2.2 ION/ION REACTIONS IN LINEAR ION TRAPS (LITs) Linear or two-dimensional (2D) quadrupole ion traps have been adapted widely for ion storage in systems comprised of Fourier transform ion cyclotron resonance (FT-ICR) spectrometers [43–45], time-of-light (TOF) [46–48], and standard 3D ion trap mass spectrometers [49,50] due to the improved trapping eficiency of the linear ion trap (LIT) and its increased ion capacity relative to a 3D ion trap. With the development of the LIT as a stand-alone mass spectrometer [51,52], interest has been directed to the implementation of ion/ion reactions in such instruments. However, a LIT that uses DC trapping voltages at the ends of the multi-pole array, which is the usual way of operating a LIT, is not suitable for simultaneous trapping of ions of both polarities, which is required in an ion/ion reaction implemented in the mutual storage mode. Methods have been developed to provide mutual ion storage of both polarities in the axial direction of the LIT by creating RF barriers at the two ends of the LIT quadrupole array. A straightforward approach is to apply an auxiliary RF voltage on the two containment lenses of the LIT, which produces the required RF barriers for ions of both polarities in the axial direction. This method has been demonstrated [32] by the Hunt group on a modiied LIT mass spectrometer (Finnigan LTQ™ mass spectrometer, Thermo Electron, San Jose, CA, USA), as shown in Figure 1.3, in the irst study of ion/ion ETD. An alternative approach to create an RF barrier to effect mutual ion storage along the axial direction is to unbalance the RF potential applied to the LIT quadrupole array by subtracting a fraction of RF amplitude applied to one set of opposing rods and adding the same amount of RF to other pair of rods [53,54]. The degree of RF unbalance is determined by the fraction of the RF amplitude subtracted from one opposing set of rods and applied to the other. When the quadrupole array and the containment lenses share the same DC offset level, unbalancing the RF on the rods creates an oscillating RF in the axial direction in the fringing region of the rod set, which is equivalent to applying RF to the containment lenses. This approach has been applied successfully to the third quadrupole (Q3) LIT of a modiied hybrid triple quadrupole/LIT (Q-Trap 2000, Applied Biosystems/MDS Sciex, Concord, ON, Canada) [54]. However, lexible control (turning ‘On’ and ‘Off’) of the RF barrier created by the unbalance of the RF is not straightforward to achieve in an MSn experiment. As a result, ion injection into the LIT and the performance of mass analysis by massselective axial ejection (MSAE) [55] may be affected adversely when the existing RF barrier at the end of the rod set cannot be applied or removed in a timely manner. To address this issue, subsequent work on the same apparatus involved superposing to the containment lenses of both collision cell (Q2) and Q3 LIT an auxiliary RF, as indicated in Figure 1.4, which can be controlled readily by the scan function during an experiment. Various ion/ion reactions have been demonstrated successfully with this setup in both the Q2 and Q3 linearion traps (LITs) with a much higher (roughly one order

10

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a)

Front

Center

Back

ESI

CI

+

–

Front lens

Back lens

(b)

0V Precursor ions moved to front section +

–10 V

(c) +5 V

Anion injection

–

+ (d) +5 V –

+

–

0V

–

(e) Ion/ion reaction

+ 0V –

(f ) –

End reaction and scan out +

–

0V

FIGURE 1.3 Schematic of a LIT instrument and experimental steps involved in ion/ion reactions (a) Injection of cations from ESI. (b) Transfer of cations to the front section of LIT. (c) Injection of reagent anions to the center section of LIT from a chemical ionization source. (d) Isolation of positively charged precursor ions and reacting reagent anions by a broadband signal. (e) Mutual storage of both polarities in the center section of LIT. (f) Removal of anions leaving the positively charge product ions for subsequent mass analysis. (ions of opposite polarities are injected along the axial direction from either end of the LIT). (Reproduced from Syka, J.E.P.; Coon, J.J.; Schroeder, M.J.; Shabanowitz, J.; Hunt, D.F., Proc. Natl. Acad. Sci. USA. 2004, 101, 9528–9533. With permission from National Academy of Sciences.)

of magnitude) reaction rate observed in the former, presumably due to the higher bath gas pressure in Q2 (ca 5 mTorr in Q2 and ca 5 × 10−5 Torr in Q3) leading to better ion cloud overlap and reduced relative velocities. Different schemes have been used for the introduction of oppositely-charged ions into the LIT. In the modiied LTQ instrument, shown schematically in Figure 1.3,

11

Ion/Ion Reactions in Electrodynamic Ion Traps Curtain plate Orifice

Fused silica capillary

Skimmer ~

Capillary holder Q0

Nano-ESI tip

Q1

~

Q2

IQ2

Q3

IQ3

Triggered +/– HV

Triggered –/+ HV

FIGURE 1.4 Schematic of a triple quadrupole/LIT mass spectrometer modiied by superposing an auxiliary RF on IQ2 and IQ3 containment lenses. (Note the pulsed dual ESI source is also illustrated.) (Reproduced from Xia, Y.; Liang, X.R.; McLuckey, S.A., J. Am. Soc. Mass Spectrom. 2005, 16, 1750–1756. With permission from Elsevier.)

positive ions formed from ESI are introduced from one end of the LIT and the negative ions produced from the chemical ionization source are admitted from the other end of the LIT [32]. Mass analysis via radial ion ejection [51] through slots in one pair of opposing rods facilitates this arrangement because ion detection occurs in the x or y-plane providing ready access to both ends of the LIT for ion sources. This arrangement for ion admission of both polarities along the z-axis of the LIT from the front and rear ends of the instrument leads to high ion injection and trapping eficiencies. Distinct from the LTQ LIT, LITs such as those marketed by ABI and MDS Sciex employ mass-selective axial ejection for mass analysis, which requires that a detector be placed near one end of the quadrupole array [52]. As a result, only one end of the LIT can be used for ion admission along the z-axis of the instrument. Because of this constraint, efforts have been made to deliver ions of both polarities from the same end of the instrument to gain the advantages associated with ion injection along the z-axis of the instrument. However, it is noteworthy that proton transfer ion/ion reactions have been implemented [56] in the same instrument by introducing multiply-charged positive ions from the front end of the instrument and admitting singly-charged negative ions from the side of the Q3 quadrupole coupled with an ASGDI source, but with lower ion injection eficiency and higher ion fragmentation due to the RF ield along the radial direction of the Q3 LIT. The irst demonstration of ion injection of both polarities via a common atmosphere/vacuum interface of an LIT employed sonic spray ionization (SSI) [57–59] to produce from the same solution both positive ions and negative ions, which were injected sequentially into the LIT [60]. Despite its relative simplicity and effectiveness for implementing a variety

12

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

of ion/ion reactions, SSI usually gives lower ionization eficiency relative to other spray methods; in addition, deleterious matrix effects on the analyte ions associated with the addition of the species intended to provide the reagent ions may become severe. The limitations associated with SSI can be largely overcome by the use of a pulsed dual polarity ionization source that allows for independent optimization of the ionization conditions for each ion polarity. The pulsed dual ESI (see Figure 1.4) and ESI/APCI sources were developed subsequently for such purposes [61,62]. High reproducibility of ion signals of both polarities and minimum interference between the two ion sources were noted when the two alternately-pulsed ion sources were placed in front of the same atmospheric/vacuum interface. Almost all types of ion/ ion reactions demonstrated previously can be accomplished using the dual ESI and ESI/APCI sources, which include reactions involving single proton transfer, single electron transfer, multiple proton transfer, etc. [61,62] It can be inferred readily from the implementation of the pulsed dual source that more than two emitters can be adapted to an LIT by sharing the common atmospheric/vacuum interface and ion path, provided that each emitter can be controlled independently by the instrument software. For example, a pulsed triple ionization source has been demonstrated successfully [63] for a sequential ion/ion reaction where two different reagents were desired. Compared to SSI, pulsed double and triple ionization sources give higher ionization eficiency and enable a wider choice for ion/ion reaction combinations, which is critical for the exploration of a broad range of ion/ion chemistries. In addition to mutual storage ion/ion reactions, transmission mode ion/ion reactions have been demonstrated also in the LIT as an alternative option, in which reactions occur when ions of one polarity pass through the quadrupole array with relatively low kinetic energy while ions of the other polarity are stored in the LIT using DC potentials applied to the containment lenses [56,64–67]. The effectiveness of the transmission mode reaction is attributed largely to the high ion injection/ transmission eficiency along the axial direction of the LIT and the fast kinetics associated with ion/ion reactions. The extent to which a reaction can proceed is determined by a number of factors such as the number density of the pre-stored ions, the kinetic energy, and the transmission time of the transmitted ion, etc. A variety of ion/ion reaction phenomena have been demonstrated in transmission mode, which include proton transfer, charge inversion, and electron transfer on both a triple quadrupole/LIT and a hybrid LIT/TOF instrument [56,64–67]. The results show that the extent of ion/ion reaction is similar when either polarity is trapped while passing the other, and the extent can be comparable also to that obtained with a mutual storage ion/ion reaction. Because of the omission of a mutual storage period, transmission mode ion/ion reactions can enjoy the advantage of improved instrument duty cycle [67]. However, the biggest advantage associated with the transmission mode approach is that minimal instrument modiication is required for the implementation of ion/ion reactions in most commercial instruments. With the use of an LIT as a reaction vessel, transmission mode ion/ion reactions are, in principle, available on any hybrid instrument such as quadrupole/TOF, LIT/Orbitrap, and LIT/FT-ICR, provided the instrument control software can execute an ion/ion reaction experiment.

13

Ion/Ion Reactions in Electrodynamic Ion Traps

1.2.3 ION/ION REACTIONS IN HYBRID INSTRUMENTS Various hybrid tandem mass spectrometers, which combine two or more distinct types of mass analyzers, have been developed to maximize analytical performance and functionality. From the standpoint of ion/ion reactions, the incorporation of an electrodynamic ion trap into a hybrid instrument allows for the physical separation of the three basic steps involved in an ion/ion reaction experiment, that is, ionization, ion/ion reaction, and mass analysis of reaction products. The separation of these processes provides for the highest degree of lexibility and minimal compromises in the optimization of each step. To date, three major types of hybrid instruments have been described for ion/ion reaction studies using an electrodynamic ion trap as the reaction vessel. The three major types of hybrid instruments are: (i) quadrupole/TOF tandem mass spectrometer; (ii) Orbitrap*; and (iii) LIT /FT-ICR. The irst type is a modiied commercial quadrupole/TOF tandem mass spectrometer (QSTAR XL, Applied Biosystems/MDS Sciex, Concord, ON, Canada) [68] which consists of three quadrupoles (ion guide (Q0), mass ilter (Q1), and Q2), and an orthogonal relectron TOF mass analyzer, as shown schematically in Figure 1.5. Although, transmission mode ion/ion reactions [66,67] were demonstrated successfully later on this instrument, ion/ion reactions were implemented initially in the mutual storage Aux. RF OR

~

SK

GR

~

IQ1

+’ve nano–ESI Q0

Q1

–’ve ESI/APCI

TOF

Q2

IQ2

IQ3

+1500 V +18 V

Cation injection

+20 V

+ 13 V +8 V

Anion injection

–14 V

–6 V

–8 V

Rf barrier

–2500 V Rf barrier

Mutual storage Release to TOF

0V

–8 V

0V

+8 V

–8 V

Rf barrier

FIGURE 1.5 Schematic of a modiied hybrid quadrupole/TOF mass spectrometer (QSTAR XL) and scan functions in a typical mutual storage mode ion/ion reaction (ions of opposite polarities are generated from a pulsed dual ion source). (Reproduced from Xia, Y.; Chrisman, P.A.; Erickson, D.E.; Liu, J.; Liang, X.R.; Londry, F.A.; Yang, M.J.; McLuckey, S.A., Anal. Chem. 2006, 78, 4146–4154. With permission from American Chemical Society.) * See Volume 4, Chapter 3: Theory and Practice of the Orbitrap™ Mass Analyzer by Alexander Makarov.

14

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

mode with the superposition of auxiliary RF signals on the two containment lenses of the Q2 quadrupole array. Compared to mass measurement with an electrodynamic ion trap, the use of a TOF mass analyzer provides superior mass analysis igure-of-merits in several respects; for example, a mass-resolving power of ca 8000 (M/ΔM FWHM) was obtained over a wide range of mass-to-charge ratios with a mass accuracy of ca 20 ppm for external calibration and 5 ppm for internal calibration [68]. Furthermore, an upper m/z limit of about 66,000 Th was observed, which is likely to be determined as much by the eficiency of the detector as it is by the ability of the LIT to store ions of such high m/z-value. A recent demonstration of bi-directional ion transfer between the three quadrupole arrays (that is, Q0, Q1, and Q2) has expanded greatly the MSn functionality of this platform, which allows for ready implementation of multi-step experiments including sequential ion/ion reactions [69]. Essentially all of the key ion/ ion reactions demonstrated in ion traps have been implemented readily on this instrument by use of a pulsed dual ion source. An example of a typical sequence of steps used in a mutual storage mode ion/ion reaction experiment with this instrument is shown in Figure 1.5. The second type, the LTQ Orbitrap XL, is the irst commercial hybrid instrument designed with ion/ion reaction capabilities as a result of the efforts of Thermo Scientiic to accommodate ETD experiments. The LTQ Orbitrap XL is comprised of an LIT, serving as both an ion/ion reaction vessel and mass analyzer, an Orbitrap [70,71] for high resolution mass analysis, and a ‘C-trap’ for ion transfer from the LIT to the Orbitrap; the ‘C-trap’ is an ion trapping device used to couple the LIT with the Orbitrap. The ion/ion ETD reaction is implemented in mutual storage mode with the positive analyte ions being produced via ESI from the front end of the instrument while the negative ions are formed by a chemical ionization source located at the rear end of the instrument. However, the irst ETD experiment demonstrated on a hybrid LTQ Orbitrap used an earlier model of the LTQ Orbitrap XL that lacked the chemical ionization module [72,73]. The key hardware modiications were the application of an auxiliary RF trapping signal to the end lenses of the LIT for mutual ion storage and the adaptation of a pulsed dual ESI source to generate both positive analyte ions and negative ETD reagent ions [74] from the inlet of the instrument [75]. The ion/ ion reaction took place in the LIT and products were transferred via the ‘C-trap’ to the Orbitrap for mass analysis, which provided a mass-resolving power of ca 60,000 and a mass accuracy within 2 ppm. This instrument was used to demonstrate protein identiication with high conidence due to both high sequence coverage (from the ion/ ion reaction) and high mass measurement accuracy (from the mass analyzer) [75]. A recent implementation of ETD reactions on the third type of hybrid instrument, a hybrid LIT/FT-ICR instrument [76], presents another example of the value in bringing high mass accuracy and mass-resolving power to the measurement of ion/ion reaction products. The hybrid instrument used a hexapole LIT as the reaction vessel by the superposition of auxiliary RF signals to the end lenses to effect mutual storage ion/ion reactions, the products of which were sent directly to the adjacent FT-ICR for mass analysis. The arrangement of the positive and negative ions sources is very similar to that for the Bruker Daltonics HCTultra™ post-translational modiication (PTM) ion trap mass spectrometer [42] described in the previous section, which introduces positive ions formed by ESI from the front end of the instrument and

Ion/Ion Reactions in Electrodynamic Ion Traps

15

admits the negative ions from a negative chemical ionization source through the side of an octopole ion guide. The advantages associated with the coupling of an FT-ICR with a LIT for the ion/ion reaction were clear, as indicated by the resolving power of 60,000 and average mass accuracy of 1.36 ppm observed in the analysis of a small (about 10 kDa) protein by ETD with the use of a relatively low magnetic ield strength (3T) cryo-magnet [76]. In summary, with the separation of the various components of an ion/ion reaction experiment, hybrid instruments provide potentially extremely powerful platforms for the gas phase analysis of ion–ion reaction products with high sensitivity, mass resolution, and mass accuracy.

1.3 METHODOLOGIES/APPLICATIONS Mass spectrometry applications that employ ion/ion chemistry have expanded rapidly in the past decade, particularly in biomolecule analysis. Ion/ion proton transfer reactions, for example, have been used widely for complex mixture analysis [12,26,77,78], ion concentration and puriication [79–82], precursor ion charge-state manipulation [82–86], simpliication of product ion mass spectra [76,81,83,88–93], etc. Charge inversion by ion/ion reactions enable conversion of ions of one polarity to the other and, with two sequential charge inversion reactions, are capable of increasing the net charge of an ion in the mass spectrometer [94–97]. In addition, metal–ion transfer between metal-containing reagents and biomolecules has been demonstrated both for polypeptides and for oligonucleotides [35,98–102]. Subsequent dissociation of the metal-adducted ions has been shown to be able to provide structural information that complements that derived from the dissociation of ions from the same analytes devoid of metals. As a unique dissociation tool using ion/ion chemistry, ETD plays a critical role in providing sequence information complementary to that obtained from other techniques and inds increasing use in peptide and protein characterization [32,33,103–111].

1.3.1 CHARGE STATE MANIPULATION: PROTON TRANSFER 1.3.1.1 Macromolecule Mixture Analysis The analysis of macromolecules by mass spectrometry was facilitated by new ionization methods such as ESI. The multiple-charging phenomenon associated with ESI, however, often leads to a mass spectrum of limited value due to the possibility of severe peak overlap when complex mixtures are analyzed. This problem can be ameliorated to a large extent by use of an ion/ion proton transfer reaction to reduce the number of charges on the ions, which enhances signiicantly the informing power [112] of the approach by distributing the charge states over a wider range of mass space (provided that such a mass range can be accommodated) and reducing the number of peaks in the spectrum. When the charge states of all analyte ions are reduced largely either to + 1 or −1, the analyte molecular weights can be obtained directly from the mass spectrum, which resembles one obtained with Matrix-assisted laser desorption/ionization. Application of this approach to the analysis of protein mixtures was demonstrated in the early studies of ion/ion reactions using both an ion trap and an atmospheric pressure reactor external to a mass spectrometer [12,77].

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

Analogous applications to nucleic acid mixture analysis have been described also [26]. The charge-squared dependence of the ion/ion reaction kinetics is of particular importance in this application as it gives rise to a desirable situation where analyte ions of various initial charge states can be reduced to largely singly-charged states with relatively little differential neutralization within the same reaction time window. Furthermore, the minimal degree of complex formation and negligible fragmentation observed commonly in proton transfer reactions contribute to the utility of this approach, which would otherwise add another factor of complexity to the analysis. 1.3.1.2 Precursor Ion Charge State Manipulation The charge state of an ion plays a central role in its chemistry, particularly in its gas phase dissociation [83,87–91,113]. For example, a general tendency has been noted in the fragmentation of protein ions that a high-charge state ion fragments preferentially at the N-terminal side of proline residues while the low-charge state gives products predominantly from C-terminal aspartic acid cleavages [83–85,87]. The chargedependent dissociation behavior of macromolecules makes desirable the ability to manipulate precursor ion charge states in bio-ion primary structure elucidation. A number of approaches are available for charge manipulation of multiply-charged positive ions, including the tuning of solution and electrospray interface conditions [114], and the use of gas-phase ion/molecule proton transfer reactions with strong bases [115–118]; however, ion/ion reactions in an electrodynamic ion trap provide the most comprehensive means for charge reduction of multiply-charged precursor ions. The high exothermicity associated with ion/ion reactions allows for an arbitrary degree of charge state reduction (including reduction to charge states not formed directly via ESI) with high eficiency through lexible control of the ion number densities in the ion trap and a well-deined reaction time window. The ability to accelerate selectively ions of particular mass-to-charge values in an electrodynamic ion trap can be used to inhibit selectively the ion/ion reaction rate of a speciic ion, which allows for a gas-phase ion concentration and puriication technique called ‘ion parking’ [79]. Ion parking can be effected by use of a low amplitude supplementary RF signal corresponding to the secular frequency of the ion of interest to accelerate the ion to a small degree so that the relative velocity between the two reactant ions is increased and their spatial overlap is decreased, leading to a reduced reaction rate. This phenomenon allows for gas-phase puriication of a protein from a protein mixture simultaneously with concentration of the overall signal from highercharge states into a parked lower-charge state that can be interrogated further via tandem mass spectrometry (MS/MS) [81]. A demonstration of gas-phase ion concentration using ion parking is illustrated in Figure 1.6, where a distribution of charge states of bovine serum albumin is concentrated largely into a single-charge state. Application of this technique to protein analysis and unknown protein identiication in a complex mixture derived from a whole cell lysate faction of Escherichia coli was described as an early example [80]. In addition to single frequency ion parking for ions within a narrow m/z-range, ion/ion reactions can be inhibited simultaneously for ions over a wider range, a phenomenon called ‘parallel ion parking,’ which can be effected by use of a tailored waveform [119], a single frequency alternating current (AC) signal of relatively high amplitude (a technique termed ‘high amplitude low

17

Ion/Ion Reactions in Electrodynamic Ion Traps (a)

(b)

Abundance

7500 500

[M+47H]47+ [M+38H]38+ [M+54H]54+ 1000

1500

2500

[M+25H]25+

12500 500

2000

1000

1500

2000

3000

3500

[M+22H]22+

4000

[M+17H]17+

2500

3000

3500

4000

2500

3000

3500

4000

(c) [M+34H]34+

Abundance

150000

500

1000

1500

2000 m/z

FIGURE 1.6 Gas-phase concentration of bovine serum albumin. Mass spectra derived: (a) from native ESI; (b) after partial proton transfer reactions and in the absence of ion parking mode; and (c) after partial proton transfer reactions with ion parking mode. (Reproduced from Reid, G.E.; Wells, J.M.; Badman, E.R.; McLuckey, S.A., Int. J. Mass Spectrom. 2003, 222, 243–258. With permission from Elsevier.)

frequency’ (HALF) parallel ion parking) [120], or a dipolar DC potential across the end-cap electrodes of a 3D ion trap [121,122]. Examples of applications using parallel ion parking include the inhibition of sequential ion/ion reactions involving irst generation products in an ETD experiment [119] and simultaneous gas-phase ion concentration for all protein components in a protein mixture [120]. 1.3.1.3 Simplification of Product Ion Mass Spectra A product ion mass spectrum from the dissociation of multiply-charged precursor ions consists generally of a mixture of fragment ions with various charge states up to that of the precursor ion. There is a general tendency for product ions derived from highly-charged precursor ions to be concentrated in a narrow mass-to-charge region that surrounds the precursor ion, leading to a mass spectrum of limited value with signiicant complexity and congestion due to severe peak overlap. Ion/ion proton transfer reactions have proven to be very useful for the simpliication of such mass spectra by reducing product ions to largely singly-charged ions and separating the overlapped peaks of different masses and charges but similar mass-to-charge ratios. This approach

18

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

(a) Pre-ion/ion rxn [M+19H]19+ (x3)

Abundance

400 300 200 100 500

700

900

1100

1300 m/z

1500

1700

1900

(b) Post-ion/ion rxn 50

b88+ b89+– + V/A b97

Abundance

40

9000

20 10

b111+ y129+ L/G y2382+ L/G

+

b100 A/Q

b86+ D/D

30

2+ b104+– b110+ [M+2H]

b103+ V/T

10000

11000

12000

13000

b130+– y136+

y137+ V/T

y143+– y151+

15000

16000

17000

[M+3H]3+

y238+ y228+ V/G W/P

y174+ D/G

2+

7000 9000

[M+H]+

y154+ D/D

y140+ A/Q

14000

y152+ V/A

11000

13000

15000

17000 19000 m/z

21000

23000

25000

27000

FIGURE 1.7 Product ion mass spectra of [M + 19H]19 + porcine elastase derived from: (a) CID of [M + 19H]19 + ; and (b) post-ion/ion proton transfer reactions. (Reproduced from Hogan, J.M.; McLuckey, S.A., J. Mass Spectrom. 2003, 38, 245–256. With permission from John Wiley & Sons Limited.)

has been used in a variety of ‘top-down’ studies of model proteins [83,86,89–91,113], and in the identiication of a priori unknown proteins in complex mixtures using database searches against in silico product ion mass spectra predicted from whole proteins [80,81]. An example of product ion manipulation using ion/ion proton transfer reactions is shown in Figure 1.7, where isolated porcine elastase [M + 19H]19 +  ions were subjected to collision-induced dissociation (CID) (Figure 1.7a) followed by an ion/ion reaction to reduce the entire product ion population of multiply-charged ions to largely singly-charged ions (Figure 1.7b). The congestion of the product ion mass spectrum in Figure 1.7a precluded conident interpretation. However, simpliication of the product ion spectrum with ion/ion reactions allowed fragment ions to be assigned without charge state ambiguity. ‘Top-down’ protein identiication via either CID or ETD followed by the examination of low mass product ions has been demonstrated also on instruments with moderate upper mass-to-charge limits (m/z = 2000–4000 Th) after all the product ions were reduced to largely singly-charged states [92,93,123]. It is noteworthy that in the latter case, the reagent ions for ETD and the reagent ions for subsequent proton transfer were both derived from the same neutral precursor species [76].

Ion/Ion Reactions in Electrodynamic Ion Traps

19

1.3.2 CHARGE INVERSION Sequential proton transfer between multiply-charged ions of one polarity and a singlycharged ion of opposite polarity often leads to stepwise charge reduction until complete ion neutralization. However, multiple proton transfer within a single encounter can produce ions of opposite polarity, a phenomenon referred to as ‘charge inversion’ [94–97]. Charge inversion enables ion formation in one polarity and analysis in the opposite polarity mode. This capability is an example of how ion/ion reactions allow for the separation of the ionization step from the ion interrogation step in that ions can be formed in one polarity, converted to the opposite polarity, and subjected to dissociation for structure determination. An example of this type of process was demonstrated in a recent ETD study of a phosphopeptide in a simple peptide mixture. Hardly any positive ion signal was observed from the phosphopeptide under positive ESI conditions; however, deprotonated phosphopeptide, which was formed in high abundance via negative ESI, yielded abundant doubly-protonated phosphopeptide ions when the mixture solution was sprayed in the negative mode followed by charge inversion of the deprotonated phosphopeptide [97]. Both positive-to-negative and negative-to-positive charge inversion reactions have been effected by use of a variety of reagents including dendrimers, proteins, and small organic compounds, with dendrimers being the most widely used. In addition to polarity switching via a single charge inversion, two consecutive charge inversion reactions can result in a net increase in ion charge. Charge increase is desirable in many instances; for example, ions of higher charge often give better detector response and allow measurement to be made with higher resolution (for example, with an FT-ICR, for which mass resolution is inversely proportional to the m/z-value of the ion). Also, the ability to access a range of charge states opens the opportunity for the study of a wide range of chemistries. In a typical experiment for charge increase using positive ions as an example, the singly-charged analyte cation is allowed initially to react with multiply-charged reagent ions of opposite polarity, which leads to the charge inversion of the analyte ion to a negative ion (Equation 1.1). In the second charge inversion step, the net effect of charge increase can be achieved when the charge inverted ion, that is, the negative analyte ion, from the previous step reacts with another multiply-charged reagent ion of different polarity (Equation 1.2). [ M + H]+ + [ N − nH]n− → [ M − H]− + [ N − (n − 2) H]( n− 2)−

(1.1)

[ M − H]− + [R + mH]m+ → [ M + 2H]2+ + [R + (m − 3)H]( m−3)+

(1.2)

1.3.3 METAL–ION TRANSFER The ability to insert selectively metal cations into macromolecule ions in the gas phase is attractive both for the study of the intrinsic interactions of metal ions with macromolecules and for maximizing the structural information available from tandem mass spectrometry. For example, singly-charged, singly-sodiated peptide cations have been shown to fragment next to the C-terminal residue [124], whereas the protonated version fragments at various locations along the peptide backbone. The insertion

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a) Abundance

[M+2H]2+ 3e4 [M+3H]3+

[M–2H+2ca]2+

2e4 1e4

500

850 m/z

1200

(b) Abundance

[M+3H]3+ 3e4 2e4

[M+H+Ag]2+

1e4

[M+2H]2+

500

[M+2Ag]2+

850 m/z

1200

Abundance

(c) 3e4 2e4 1e4

500

[M+3H]3+ [M+Ni]2+ [M+2H]2+ [M–2H+2Ni]2+ 850 m/z

1200

FIGURE 1.8 Metal-ion transfer via cation switching ion/ion reactions of Trp-11 neurotensin with: (a) calcium acetate anions; (b) silver nitrate anions; and (c) nickel acetate anions. (Reproduced from Newton, K.A.; McLuckey, S.A., J. Am. Chem. Soc. 2003, 125, 12404–12405. With permission from American Chemical Society.)

of various metal ions into peptide or protein ions has been accomplished in the gas phase by cation-switching reactions involving multiply-protonated polypeptides and metal-containing anions, as illustrated in Figure 1.8, where calcium, silver, and nickelcontaining trp-11 neurotensin were formed in the gas phase [98]. The selective removal of cations, either protons or metal ions, from peptide or protein ions may be desirable also in some cases and a number of examples of ion/ion reactions for this purpose have been discussed [99,100]. In addition to metal transfer into polypeptide ions, transition metal ion insertion into oligodeoxynucleotide anions has been effected in the gas phase via ion/ion reactions of transition metal complex cations with multiply-charged oligodeoxynucleotide anions. Depending upon the metal, ligands, and reactant ion charge states, metal transfer to the oligodeoxynucleotide anion competes more or less effectively with other processes, such as electron transfer and cation/anion complex formation [102]. Gas-phase metal-ion transfer to macromolecules via ion/ion reactions

Ion/Ion Reactions in Electrodynamic Ion Traps

21

enables the separation of the ionization process from the formation of metal-containing macromolecule ions, which allows the optimization of both processes individually and the production of metal-containing species not formed readily in solution.

1.3.4 ELECTRON TRANSFER DISSOCIATION (ETD) Although the irst bio-ion/ion electron transfer reaction involved multiply-deprotonated oligonucleotides in reaction with xenon cations [125,126] (later work was extended to those involving multiply-deprotonated peptides [127]), extensive applications of ETD have been focused largely on the reaction of multiply-protonated polypeptides with radical anions, due to the unique sequence information provided by this reactive combination. When an electron is transferred from an anion to a multiply-charged polypeptide cation, an open-shell radical cation is created, which fragments subsequently to produce c- and z-type sequence ions from backbone N−Cα bond cleavages, a phenomenon highly analogous to ECD [128,129]. Compared to traditional activation methods such as CID, ETD tends to be less selective than CID in terms of the range of structurally-informative channels that contribute to the product ion mass spectrum and, as a result, tends to provide more extensive sequence information. In addition, ETD tends not to cleave bonds to PTM that are relatively labile under CID conditions. Both features of ETD have been noted in the irst ETD study on multiply-protonated polypeptides, as shown in Figure 1.9, in which a methyl-esteriied phosphopeptide generated from a tryptic digest of human nuclear proteins was subjected to dissociation by both CID and ETD [32]. The CID product ion mass spectrum in Figure 1.9a is dominated by product ions corresponding to neutral losses of phosphoric acid and either water or methanol and yields little, if any, sequence ion information, whereas complete sequence coverage is achieved in the ETD spectrum, Figure 1.9b, with 13 out of 14 possible c/z-type ions observed. The propensity of ETD to retain the PTM information is demonstrated also in Figure 1.9b where no fragment ions are observed corresponding to the loss of phosphoric acid. Although Figure 1.9 illustrates an extreme case where essentially no sequence information can be acquired from CID, there is a consensus that, in general, ETD gives complementary sequence information to that provided by other activation methods, such as CID, in either modiied or unmodiied peptides [67,106,130,131]. This conclusion has been supported widely by many studies, as exempliied by a recent report that described a rapid alternating transmission mode ETD and CID for the characterization of polypeptides ions, including a phosphopeptide, a glycopeptide, and an unmodiied tryptic peptide [67]. In the glycopeptide (Figure 1.10), for instance, ETD (Figure 1.10a) generates information about both peptide sequence and locations of the glycosylation sites, whereas CID (Figure 1.10b) provides information about the glycan structure. Motivated by the complementary nature of ETD, combined techniques involving ETD, for example CID coupled with ETD, have been used for peptide unknown protein database search with improved performance [106,132]. In addition to phosphorylation and glycosylation, disulide linkage formation is another PTM that can be characterized more readily by ETD than by excitation with either photons or low-energy collisions, the latter of which tend not to cleave disulide bonds present in multiply-protonated polypeptides [133,134]. The rupture

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a) 144 300

b

E

CID

R E R pS L pS 1234 1091 935 768 655 488 332 189

y

(M+3H–H3PO4–MeOH)+++

100 Relative abundance

467 580 747 903 1046 1234

R

(M+3H–2H3PO4)+++

Int: 2.08 × 108

(M+3H–H3PO4–H2O)+++ (M+3H–H3PO4)+++

50

0 (b) c z

Relative abundance

484 597 764 920 1063 1234 R E R pS L pS

161 317 E R

1234 1075 919 752 639 (M+3H)+++

14 z2

10

472 316 173

(M+2H)++

c6

c4 z6

z1 c3

2

y1 200

z3

a2 400

z5

z4

z6**

(M+1H)+

Int: 2.19 × 105

c2 6

ETD

c7 z7

y6 a7

600

800

1000

1200

m/z

FIGURE 1.9 Comparison of mass spectra derived from: (a) CID; and (b) ETD of a phosphopeptide. (Reproduced from Syka, J.E.P.; Coon, J.J.; Schroeder, M.J.; Shabanowitz, J.; Hunt, D.F., Proc. Natl. Acad. Sci. USA. 2004, 101, 9528–9533. With permission from National Academy of Sciences.)

of disulide linkages using ETD is particularly useful in the characterization of polypeptides with internal disulide linkages due to the fact that the sequence information within the cyclic structure formed by the disulide bond is, in most cases, accessible only when the ring structure is opened [135]. In addition to the primary structure information obtained from backbone cleavage, ETD allows also for a ready differentiation of some isomeric structures of the protein side-chain, for example, isoaspartic acid and aspartic acid residues, because its characteristic dissociation of N−Cα bonds leads to the formation of diagnostic ions [136]. Besides the structural information provided for polypeptides, ETD has been shown to be useful for the characterization of glycerophosphocholine lipids [137] and may also play an important role in quantitation of peptides and proteins. The variety of applications related to ETD is expected to increase rapidly as its compatibility with high throughput experiments is

c3

z3

c4

600

c5

c6

c7 c8 c9

1100

c10

c11 c12

c13

2+

1600

z4 8

9

Man

Man

GlcNAc

GlcNAc

2100

2600

Activation on 2+: 48.60 kHz, 1.0 Vpp z6 z14 z12 z10 z 5 z7 z z13 z11 z

Xyl

Fuc

Man

SKP A Q G Y G Y L G V F N NSK

3100

0 700

800

[M-FucXylMan3+3H]3+

900

1000

1100 m/z

1200

-Man

1300

-Man

1400

-Xyl

1500

SKP A Q G Y G Y L G V F N NSK [M-Xyl+3H]3+ [M-Fuc+3H]3+ Fuc GlcNAc [M-Man+3H]3+ [M-FucXylMan GlcNAc+2H]2+ 3 2+ [M-FucXylMan3GlcNAc2+2H] GlcNAc [M-XylMan3GlcNAc+2H]2+ 4.0E3 [M-FucXyl+3H]3+ 3+ 2+ Xyl Man Man [M-FucXylMan3+2H] [M-XylMan+3H]3+ [M-FucXylMan2+2H]2+ [M-FucMan+3H]3+ Man 2+ [M-FucXylMan+3H]3+ [M-FucXylMan+2H]2+ [M-Fuc+2H] -GlcNAc 2.0E3 -Man -GlcNAc [M-FucXyl+2H]2+ [M-FucXylMan2+3H]3+

6.0E3

0 100

40

80

120

160

3+ x 22.9

Neutral losses

Abundance (arb. unit)

FIGURE 1.10 Product ion mass spectra of a tryptic lectin glycopeptide derived from: (a) transmission mode ETD of the triply-charged peptide with azobenzene radical anions; and (b) beam-type CID of the triply-charged peptide. (Reproduced from Han, H.; Xia, Y.; Yang, M.; McLuckey, S.A., Anal. Chem. 2008, 80, 3492–3497. With permission from American Chemical Society.)

(b)

(a)

Ion/Ion Reactions in Electrodynamic Ion Traps 23

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

developed. For example, implementation of ETD on the chromatographic timescale has been demonstrated already [138], and has been found to be dependent upon the high rates of reaction that can be achieved in the LIT. Because of the importance of ETD, a variety of efforts have been made for its characterization and to improve its performance. A major complication in an ETD experiment is the existence of various competing channels, which include proton transfer, side-chain losses, electron transfer without dissociation (ET, no D), and electron transfer leading to backbone cleavages (that is, ETD), with only the latter being desirable in terms of structural characterization [139]. The competing channels are affected by many factors including, but not limited to, the identity of the reagent radical anion, bath gas temperature, the cation charge identity and its location on a multiply-charged polypeptide, the peptide size, and the charge states of the polypeptide [34,131,139–142]. Recent studies have shown that an ETD reagent anion with favorable Franck–Condon factors and a relatively low electron afinity for the neutral form of the reagent tends to give higher electron transfer relative to proton transfer [141]. When the cation charge identities are considered, similar degrees of electron transfer were observed for multiply-protonated peptide ions containing protonated histidine, arginine, or lysine, which were much higher than that for peptides having ixed-charge sites. Among the four types of cation-charge sites, protonated histidine showed the highest degree of ET, no D, while no apparent intact electron transfer products were observed for peptides with protonated arginine or lysine. All cation types showed side-chain losses with arginine yielding the greatest fraction and lysine giving the smallest [142]. A general trend with increased partitioning into the ET channel was observed when ETD was performed on a polypeptide with higher charge, which was accompanied also by the decreased contribution from side-chains losses and increased partitioning into the ETD channel [139]. These results highlight the importance of using as high a charge state as possible in an ETD experiment to maximize sequence information. As a result, attention has been directed to the development of various methods to increase the charges on the peptides such as the manipulation of the solution composition of tryptic digests [143], use of other enzymatic digestions, such as endoproteinase Lys-C digestion [132], and microwave-assisted D-cleavages [144], to produce peptides of larger size which are expected to produce high charges from ESI, etc. It is noteworthy that not only the charge state but also the size of the polypeptide affects signiicantly the partitioning of the ETD channel [131,139], with larger size correlating inversely with the fraction of ETD relative to the sequence-uninformative channels. The ET, no D channel is not informative in the sense that no sequence ions are produced directly. However, the ET, no D product has been proposed to consist of c/z-fragments remaining bound via noncovalent interactions [128] or comprising of an intact protein ion with a covalent bond weakened signiicantly by the electron attachment [145]. As a result, a variety of approaches have been developed to dissociate ET, no D products to improve the production of the sequence ions [66,67,146,147].

1.4 CONCLUSIONS The robust nature of ion/ion chemistry (for example, fast kinetics due to the long range Coulombic attraction and the universal nature of ion/ion reactions because of high

Ion/Ion Reactions in Electrodynamic Ion Traps

25

exothermicities) enables implementation of ion/ion reactions under a variety of conditions; however, the combination of ion/ion reactions with electrodynamic ion traps has many advantages. The use of an electrodynamic ion trap as an ion/ion reaction vessel allows for the separation of the major components of an MS experiment, that is, ionization, reaction, and mass analysis. The MSn functionality of an ion trap enables consecutive ion/ion reactions between multiple mass selection steps and allows for the exploration of different ion/ion reaction phenomena within a single experiment. The ease with which an LIT can be coupled with other mass spectrometric components makes possible the implementation of ion/ion reactions with virtually any form of mass analyzer. In addition to the commonly-used mutual storage mode in LITs, ion/ion reactions can be implemented in transmission mode, which requires minimal hardware modiication and provides higher instrument duty cycles. The development of various new ion inlets such as pulsed dual and triple ion sources delivers high versatility to an instrument for the exploration of the high dimensionality of the ion/ion chemistry. Proton transfer ion/ion reactions have found various applications in electrodynamic ion traps including biopolymer mixture analysis by charge reduction of multiply-charged components generated by ESI to largely singly-charged ions. A similar strategy has been applied also to mixtures of product ions generated in a tandem mass spectrometric experiment to facilitate spectral interpretation. The well-deined ion frequencies established by ion storage in quadrupolar ion traps combined with ion/ion chemistry allow for ready charge reduction of a precursor ion to any arbitrary extent and gas-phase ion puriication and concentration by use of ‘ion parking’ or ‘parallel ion parking.’ Charge inversion together with both metal-ion insertion and removal provide means to convert ions from one type to another. As an important dissociation tool, ETD provides sequence information, complementary to that provided by traditional CID methods, and tends to retain PTM information during the dissociation. The unique information provided by ETD makes it a valuable tool in the study of molecular biology. The recent introduction of commercially-available instruments capable of executing ion/ion reactions in ion traps will accelerate undoubtedly the development and applications of ion/ion reactions.

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69. Xia, Y.; Thomson, B.A.; McLuckey, S.A. Bi-directional ion transfer between quadrupole arrays: MSn ion/ion reaction experiments on a quadrupole/time-of-light tandem mass spectrometer. Anal. Chem. 2007, 79, 8199–8206. 70. Hu, Q.Z.; Noll, R.J.; Li, H.Y.; Makarov, A.; Hardman, M.; Cooks, R.G. The Orbitrap: A new mass spectrometer. J. Mass Spectrom. 2005, 40, 430–443. 71. Scigelova, M.; Makarov, A. Orbitrap mass analyzer-overview and applications in proteomics. Proteomics 2006, 6, 16–21. 72. Erickson, B. Linear ion trap/Orbitrap mass spectrometer. Anal. Chem. 2006, 78, 2089–2089. 73. Makarov, A.; Denisov, E.; Kholomeev, A.; Baischun, W.; Lange, O.; Strupat, K.; Horning, S. Performance evaluation of a hybrid linear ion trap/Orbitrap mass spectrometer. Anal. Chem. 2006, 78, 2113–2120. 74. Huang, T.Y.; Emory, J.F.; O’Hair, R.A.J.; McLuckey, S.A. Electron transfer reagent anion formation via electrospray ionization and collision-induced dissociation. Anal. Chem. 2006, 78, 7387–7391. 75. McAlister, G.C.; Phanstiel, D.; Good, D.M.; Berggren, W.T.; Coon, J.J. Implementation of electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer. Anal. Chem. 2007, 79, 3525–3534. 76. Kaplan, D.A.; Hartmer, R.; Speir, J.P.; Stoermer, C.; Gumerov, D.; Easterling, M.L.; Brekenfeld, A.; Kim, T.; Laukien, F.; Park, M.A. Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer. Rapid Commun. Mass Spectrom. 2008, 22, 271–278. 77. Stephenson, J.L.; McLuckey, S.A. Ion/ion proton transfer reactions for protein mixture analysis. Anal. Chem. 1996, 68, 4026–4032. 78. VerBerkmoes, N.C.; Strader, M.B.; Smiley, R.D.; Howell, E.E.; Hurst, G.B.; Hettich, R.L.; Stephenson, J.L. Intact protein analysis for site-directed mutagenesis overexpression products: Plasmid-encoded R67 dihydrofolate reductase. Anal. Biochem. 2002, 305, 68–81. 79. McLuckey, S.A.; Reid, G.E.; Wells, J.M. Ion parking during ion/ion reactions in electrodynamic ion traps. Anal. Chem. 2002, 74, 336–346. 80. Reid, G.E.; Shang, H.; Hogan, J.M.; Lee, G.U.; McLuckey, S.A. Gas-phase concentration, puriication, and identiication of whole proteins from complex mixtures. J. Am. Chem. Soc. 2002, 124, 7353–7362. 81. Amunugama, R.; Hogan, J.M.; Newton, K.A.; McLuckey, S.A. Whole protein dissociation in a quadrupole ion trap: Identiication of an a priori unknown modiied protein. Anal. Chem. 2004, 76, 720–727. 82. He, M.; Reid, G.E.; Shang, H.; Lee, G.U.; McLuckey, S.A. Dissociation of multiple protein ion charge states following a single gas-phase puriication and concentration procedure. Anal. Chem. 2002, 74, 4653–4661. 83. Wells, J.M.; Stephenson, J.L.; McLuckey, S.A. Charge dependence of protonated insulin decompositions. Int. J. Mass Spectrom. 2000, 203, A1–A9. 84. Reid, G.E.; Wu, J.; Chrisman, P.A.; Wells, J.M.; McLuckey, S.A. Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry. Anal. Chem. 2001, 73, 3274–3281. 85. Hogan, J.M.; McLuckey, S.A. Charge state dependent collision-induced dissociation of native and reduced porcine elastase. J. Mass Spectrom. 2003, 38, 245–256. 86. Pitteri, S.J.; Chrisman, P.A.; Badman, E.R.; McLuckey, S.A. Charge-state dependent dissociation of a trypsin/inhibitor complex via ion trap collisional activation. Int. J. Mass Spectrom. 2006, 253, 147–155. 87. Mekecha, T.T.; Amunugama, R.; McLuckey, S.A. Ion trap collision-induced dissociation of human hemoglobin α-chain cations. J. Am. Soc. Mass Spectrom. 2006, 17, 923–931.

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88. Cargile, B.J.; McLuckey, S.A.; Stephenson, J.L. Identiication of bacteriophage MS2 coat protein from E-coli lysates via ion trap collisional activation of intact protein ions. Anal. Chem. 2001, 73, 1277–1285. 89. Newton, K.A.; Chrisman, P.A.; Reid, G.E.; Wells, J.M.; McLuckey, S.A. Gaseous apomyoblobin ion dissociation in a quadrupole ion trap: [M + 2H]2 + –[M + 21H]21 + . Int. J. Mass Spectrom. 2001, 212, 359–376. 90. Wells, J.M.; Reid, G.E.; Engel, B.J.; Pan, P.; McLuckey, S.A. Dissociation reactions of gaseous ferro-, ferri-, and apo-cytochrome c ions. J. Am. Soc. Mass Spectrom. 2001, 12, 873–876. 91. Engel, B.J.; Pan, P.; Reid, G.E.; Wells, J.M.; McLuckey, S.A. Charge state dependent fragmentation of gaseous protein ions in a quadrupole ion trap: Bovine ferri-, ferro-, and apo-cytochrome c. Int. J. Mass Spectrom. 2002, 219, 171–187. 92. Coon, J.J.; Ueberheide, B.; Syka, J.E.P.; Dryhurst, D.D.; Ausio, J.; Shabanowitz, J.; Hunt, D.F. Protein identiication using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 2005, 102, 9463–9468. 93. Bowers, J.J.; Liu, J.; Gunawardena, H.P.; McLuckey, S.A. Protein identiication via ion trap collision-induced dissociation and examination of low mass product ions. J. Mass Spectrom. 2008, 43, 23–34. 94. He, M.; Emory, J.F.; McLuckey, S.A. Reagent anions for charge inversion of polypeptide/ protein cations in the gas phase. Anal. Chem. 2005, 77, 3173–3182. 95. He, M.; McLuckey, S.A. Two ion/ion charge inversion steps to form a doubly protonated peptide from a singly protonated peptide in the gas phase. J. Am. Chem. Soc. 2003, 125, 7756–7757. 96. He, M.; McLuckey, S.A. Increasing the negative charge of a macroanion in the gas phase via sequential charge inversion reactions. Anal. Chem. 2004, 76, 4189–4192. 97. Gunawardena, H.P.; Emory, J.F.; McLuckey, S.A. Phosphopeptide anion characterization via sequential charge inversion and electron-transfer dissociation. Anal. Chem. 2006, 78, 3788–3793. 98. Newton, K.A.; McLuckey, S.A. Gas-phase peptide/protein cationizing agent switching via ion/ion reactions. J. Am. Chem. Soc. 2003, 125, 12404–12405. 99. Newton, K.A.; McLuckey, S.A. Generation and manipulation of sodium cationized peptides in the gas phase. J. Am. Soc. Mass Spectrom. 2004, 15, 607–615. 100. Newton, K.A.; He, M.; Amunugama, R.; McLuckey, S.A. Selective cation removal from gaseous polypeptide ions: Proton vs. sodium ion abstraction via ion/ion reactions. Phys. Chem. Chem. Phys. 2004, 6, 2710–2717. 101. Hodges, B.D.M.; Liang, X.; McLuckey, S.A. Generation of di-lithiated peptide ions from multiply protonated peptides via ion/ion reactions. Int. J. Mass Spectrom. 2007, 267, 183–189. 102. Barlow, C.K.; Hodges, B.D.M.; Xia, Y.; O’Hair, R.A.J.; McLuckey, S.A. Gas-phase ion/ ion reactions of transition metal complex cations with multiply charged oligodeoxynucleotide anions. J. Am. Soc. Mass Spectrom. 2008, 19, 281–293. 103. Chi, A.; Huttenhower, C.; Geer, L.Y.; Coon, J.J.; Syka, J.E.P.; Bai, D.L.; Shabanowitz, J.; Burke, D.J.; Troyanskaya, O.G.; Hunt, D.F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. USA 2007, 104, 2193–2198. 104. Khidekel, N.; Ficarro, S.B.; Clark, P.M.; Bryan, M.C.; Swaney, D.L.; Rexach, J.E.; Sun, Y.E.; Coon, J.J.; Peters, E.C.; Hsieh-Wilson, L.C. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nature Chem. Biol. 2007, 3, 339–348. 105. Catalina, M.I.; Koeleman, C.A.M.; Deelder, A.M.; Wuhrer, M. Electron transfer dissociation of N-glycopeptides: Loss of the entire N-glycosylated asparagine side chain. Rapid Commun. Mass Spectrom. 2007, 21, 1053–1061.

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106. Molina, H.; Horn, D.M.; Tang, N.; Mathivanan, S.; Pandey, A. Global proteomic proiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 2007, 104, 2199–2204. 107. Wuhrer, M.; Stam, J.C.; van de Geijn, F.E.; Koeleman, C.A.M.; Verrips, C.T.; Dolhain, R.J.E.M.; Hokke, C.H.; Deelder, A.M. Glycosylation proiling of immunoglobulin G (IgG) subclasses from human serum. Proteomics 2007, 7, 4070–4081. 108. Srikanth, R.; Wilson, J.; Bridgewater, J.D.; Numbers, J.R.; Lim, J.; Olbris, M.R.; Kettani, A.; Vachet, R.W. Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation. J. Am. Soc. Mass Spectrom. 2007, 18, 1499–1506. 109. Zhang, Q.B.; Tang, N.; Brock, J.W.C.; Mottaz, H.M.; Ames, J.M.; Baynes, J.W.; Smith, R.D.; Metz, T.O. Enrichment and analysis of nonenzymatically glycated peptides: Boronate afinity chromatography coupled with electron-transfer dissociation mass spectrometry. J. Proteome Res. 2007, 6, 2323–2330. 110. Zhang, Q.B.; Tang, N.; Schepmoes, A.A.; Phillips, L.S.; Smith, R.D.; Metz, T.O. Proteomic proiling of nonenzymatically glycated proteins in human plasma and erythrocyte membranes. J. Proteome Res. 2008, 7, 2025–2032. 111. Bunger, M.K.; Cargile, B.J.; Ngunjiri, A.; Bundy, J.L.; Stephenson, J.L. Automated proteomics of E. coli via top-down electron-transfer dissociation mass spectrometry. Anal. Chem. 2008, 80, 1459–1467. 112. Liu, J.; Chrisman, P.A.; Erickson, D.E.; McLuckey, S.A. Relative information content and top-down proteomics by mass spectrometry: Utility of ion/ion proton-transfer reactions in electrospray-based approaches. Anal. Chem. 2007, 79, 1073–1081. 113. Watson, D.J.; McLuckey, S.A. Charge state dependent ion trap collision-induced dissociation of reduced bovine and porcine trypsin cations. Int. J. Mass Spectrom. 2006, 255, 53–64. 114. Muddiman, D.C.; Cheng, X.H.; Udseth, H.R.; Smith, R.D. Charge-state reduction with improved signal intensity of oligonucleotides in electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 1996, 7, 697–706. 115. McLuckey, S.A.; Van Berkel, G.J.; Glish, G.L. Reactions of dimethylamine with multiply charged ions of cytochrome c. J. Am. Chem. Soc. 1990, 112, 5668–5670. 116. McLuckey, S.A.; Glish, G.L.; Van Berkel, G.J. Charge determination of product ions formed from collision-induced dissociation of multiply protonated molecules via ion molecule reactions. Anal. Chem. 1991, 63, 1971–1978. 117. Williams, E.R. Proton transfer reactivity of large multiply charged ions. J. Mass Spectrom. 1996, 31, 831–842. 118. Carr, S.R.; Cassady, C.J. Reactivity and gas-phase acidity determinations of small peptide ions consisting of 11 to 14 amino acid residues. J. Mass Spectrom. 1997, 32, 959–967. 119. Chrisman, P.A.; Pitteri, S.J.; McLuckey, S.A. Parallel ion parking: Improving conversion of parents to irst-generation products in electron transfer dissociation. Anal. Chem. 2005, 77, 3411–3414. 120. Chrisman, P.A.; Pitteri, S.J.; McLuckey, S.A. Parallel ion parking of protein mixtures. Anal. Chem. 2006, 78, 310–316. 121. Grosshans, P.B.; Ostrander, C.M.; Walla, C.A. Methods and apparatus to control charge neutralization reactions in ion traps, U.S. Patent 2003, 6,570,151, B1. 122. Grosshans, P.B.; Ostrander, C.M.; Walla, C.A. Methods and apparatus to control charge neutralization reactions in ion traps, U.S. Patent 2004, 6,674,067, B2. 123. Chi, A.; Bai, D.L.; Geer, L.Y.; Shabanowitz, J.; Hunt, D.F. Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry. Int. J. Mass Spectrom. 2007, 259, 197–203. 124. Lin, T.; Payne, A.H.; Glish, G.L. Dissociation pathways of alkali-cationized peptides: Opportunities for C-terminal peptide sequencing. J. Am. Soc. Mass Spectrom. 2001, 12, 497–504.

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125. Herron, W.J.; Goeringer, D.E.; McLuckey, S.A. Gas-phase electron-transfer reactions from multiply-charged anions to rare-gas cations. J. Am. Chem. Soc. 1995, 117, 11555–11562. 126. McLuckey, S.A.; Stephenson, J.L.; O’Hair, R.A.J. Decompositions of odd- and evenelectron anions derived from deoxy-polyadenylates. J. Am. Soc. Mass Spectrom. 1997, 8, 148–154. 127. Coon, J.J.; Shabanowitz, J.; Hunt, D.F.; Syka, J.E.P. Electron transfer dissociation of peptide anions. J. Am. Soc. Mass Spectrom. 2005, 16, 880–882. 128. Zubarev, R.A.; Kelleher, N.L.; McLafferty, F.W. Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 1998, 120, 3265–3266. 129. Zubarev, R.A. Reactions of polypeptide ions with electrons in the gas phase. Mass Spectrom. Rev. 2003, 22, 57–77. 130. Zubarev, R.A.; Zubarev, A.R.; Savitski, M.M. Electron capture/transfer versus collisionally activated/induced dissociations: Solo or duet? J. Am. Soc. Mass Spectrom. 2008, 19, 753–761. 131. Good, D.M.; Wirtala, M.; McAlister, G.C.; Coon, J.J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell Proteomics 2007, 6, 1942–1951. 132. Wu, S.L.; Huehmer, A.F.R.; Hao, Z.Q.; Karger, B.L. On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modiications. J. Proteome Res. 2007, 6, 4230–4244. 133. Chrisman, P.A.; Pitteri, S.J.; Hogan, J.M.; McLuckey, S.A. SO2– electron transfer ion/ion reactions with disulide linked polypeptide ions. J. Am. Soc. Mass Spectrom. 2005, 16, 1020–1030. 134. Gunawardena, H.P.; Gorenstein, L.; Erickson, D.E.; Xia, Y.; McLuckey, S.A. Electron transfer dissociation of multiply protonated and ixed charge disulide linked polypeptides. Int. J. Mass Spectrom. 2007, 265, 130–138. 135. Stephenson, J.L.; Cargile, B.J.; McLuckey, S.A. Ion trap collisional activation of disulide linkage intact and reduced multiply protonated polypeptides. Rapid Commun. Mass Spectrom. 1999, 13, 2040–2048. 136. O’Connor, P.B.; Cournoyer, J.J.; Pitteri, S.J.; Chrisman, P.A.; McLuckey, S.A. Differentiation of aspartic and isoaspartic acids using electron transfer dissociation. J. Am. Soc. Mass Spectrom. 2006, 17, 15–19. 137. Liang, X.L.R.; Liu, J.; Leblanc, Y.; Covey, T.; Ptak, A.C.; Brenna, J.T.; McLuckey, S.A. Electron transfer dissociation of doubly sodiated glycerophosphocholine lipids. J. Am. Soc. Mass Spectrom. 2007, 18, 1783–1788. 138. Udeshi, N.D.; Shabanowitz, J.; Hunt, D.F.; Rose, K.L. Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS. FEBS J. 2007, 274, 6269–6276. 139. Liu, J.; Huang, T.Y.; McLuckey, S.A. Charge state dependence of proton transfer versus electron transfer in a gas-phase ion/ion electron transfer dissociation process on tryptic peptides. Proc. 56th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, June 1, 2008. 140. Coon, J.J.; Syka, J.E.P.; Schwartz, J.C.; Shabanowitz, J.; Hunt, D.F. Anion dependence in the partitioning between proton and electron transfer in ion/ion reactions. Int. J. Mass Spectrom. 2004, 236, 33–42. 141. Gunawardena, H.P.; He, M.; Chrisman, P.A.; Pitteri, S.J.; Hogan, J.M.; Hodges, B.D.M.; McLuckey, S.A. Electron transfer versus proton transfer in gas-phase ion/ion reactions of polyprotonated peptides. J. Am. Chem. Soc. 2005, 127, 12627–12639. 142. Xia, Y.; Gunawardena, H.P.; Erickson, D.E.; McLuckey, S.A. Effects of cation chargesite identity and position on electron-transfer dissociation of polypeptide cations. J. Am. Chem. Soc. 2007, 129, 12232–12243.

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143. Kjeldsen, F.; Giessing, A.M.B.; Ingrell, C.R.; Jensen, O.N. Peptide sequencing and characterization of post-translational modiications by enhanced ion-charging and liquid chromatography electron-transfer dissociation tandem mass spectrometry. Anal. Chem. 2007, 79, 9243–9252. 144. Hauser, N.J.; Han, H.L.; McLuckey, S.A.; Basile, F. Electron transfer dissociation of peptides generated by microwave D-cleavage digestion of proteins. J. Proteome Res. 2008, 7, 1867–1872. 145. Turecek, F. N-Cα bond dissociation energies and kinetics in amide and peptide radicals. Is the dissociation a non-ergodic process? J. Am. Chem. Soc. 2003, 125, 5954–5963. 146. Swaney, D.L.; McAlister, G.C.; Wirtala, M.; Schwartz, J.C.; Syka, J.E.P.; Coon, J.J. Supplemental activation method for high-eficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 2007, 79, 477–485. 147. Han, H.L.; Xia, Y.; McLuckey, S.A. Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer. Rapid Commun. Mass Spectrom. 2007, 21, 1567–1573.

Hydrogen/ 2 Gas-Phase Deuterium Exchange in Quadrupole-Ion Traps Joseph E. Chipuk and Jennifer S. Brodbelt CONTENTS 2.1

Introduction .................................................................................................... 36 2.1.1 Historical Perspective ......................................................................... 36 2.1.2 Theory of Gas-Phase Hydrogen/Deuterium (H/D) Exchange Experiments ........................................................................................ 37 2.1.2.1 Deuterating Agents .............................................................. 38 2.1.2.2 Proposed Mechanisms ......................................................... 38 2.2 Practical Aspects of Gas-Phase Hydrogen/Deuterium (H/D) Exchange .......40 2.2.1 Motivation for Gas-Phase Hydrogen/Deuterium (H/D) Exchange Experiments ....................................................................... 41 2.2.2 Instrumentation for Gas-Phase Hydrogen/Deuterium (H/D) Exchange Experiments ....................................................................... 42 2.2.2.1 Ion Trapping for Gas-Phase Hydrogen/Deuterium (H/D) Exchange ................................................................... 42 2.2.2.2 Reagent Inlet Systems .......................................................... 42 2.2.3 Methods ..............................................................................................44 2.2.3.1 Typical Reaction Conditions ................................................44 2.2.3.2 Mass Spectral Interpretation ................................................ 45 2.2.3.3 Reaction Kinetics ................................................................. 45 2.3 Current Areas of Research .............................................................................46 2.3.1 Small Molecules ................................................................................. 47 2.3.1.1 Fundamental Studies of Model Compounds........................ 47 2.3.1.2 Isomer Differentiation.......................................................... 49 2.3.2 Peptides and Proteins .......................................................................... 50 2.3.2.1 Model Peptides..................................................................... 51 2.3.2.2 Proteins ................................................................................ 51 2.3.3 Nucleosides, Nucleotides, and Oligonucleotides ................................ 54 2.4 Conclusions ..................................................................................................... 55 Acknowledgment ..................................................................................................... 55 References ................................................................................................................ 56

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2.1

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

INTRODUCTION

Ever since the irst report of using hydrogen/deuterium (H/D) exchange reactions in the gas phase to count ‘active’ hydrogen atoms in organic ions [1], the potential utility of the H/D exchange method for probing structural aspects of ions has been recognized by a succession of various research groups. The H/D exchange method involves allowing a mass-selected set of ions to undergo reactions with a deuterated reagent in which labile, accessible hydrogen atoms of the analyte ions (typically surface-exposed hydrogen atoms attached to nitrogen, oxygen, or sulfur atoms) may exchange for deuterium atoms, thus causing easily monitored step-wise mass shifts in the mass spectrum. Numerous studies have shown that different active sites within ions exhibit different rates of H/D exchange, and the rates and extents of H/D exchange depend on both the intrinsic basicities (or acidities) of the reagent and analyte and the accessibilities of active hydrogen atoms. Investigations of H/D exchange reactions are a natural it for quadrupole ion trap mass spectrometers because the kinetics of reactions can be monitored accurately by variation of the ion storage time, deuterated reagents can be introduced easily and in controlled fashion to the ion trap via several alternative means, and targeted analyte ions can be mass selected readily and isolated prior to reaction. As illustrated in this chapter, applications of H/D exchange in quadrupole-ion traps have ranged from those involving small organic molecules, especially involving comparisons of isomers, to larger biological molecules for which conformational effects play a signiicant role.

2.1.1

HISTORICAL PERSPECTIVE

The origin of gas-phase H/D exchange reactions can be traced to the pioneering experiments by Hunt and co-workers who investigated gas-phase chemical ionization (CI) using D2O as a reagent gas [1]. These studies differed from modern gasphase H/D-exchange reactions in two important ways: (1) they involved the reaction of ‘ionized D2O’ (meaning the radical cation D2O + • generated by electron ionization (EI) of D2O, the CI product ion D3O +, or the CI product ion clusters D + (D2O)n +) with neutral analytes rather than the reaction of ionized analytes with a neutral deuterating agent; and (2) the CI conditions employed were more energetic than those for reactions between neutral deuterating agents and ions generated by soft ionization methods such as electrospray ionization (ESI). Nevertheless, the groundbreaking revelation came not in the eficacy of ionized D2O as an exceptional CI reagent, but rather the appearance of the isotopically-modiied mass spectra. In addition to the ionization of the neutral analyte, the observed mass spectra contained mass-shifted peaks associated with the incorporation of a deuterium atom for each of the ‘active or acidic’ hydrogen atoms, establishing a new landmark gas-phase ion/molecule reaction. Furthermore, while small amounts of deuterium exchange were observed also for ketones, aldehydes, and esters, no appreciable exchange was noted for other unsaturated organics such as benzene and stilbene. Thus, the reaction of ionized D2O was determined to be selective for particular hydrogen atoms and was deemed, therefore, to be useful in determining the types of functional groups present in a molecule known to contain speciic heteroatoms.

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37

The initial CI studies performed by Hunt and co-workers inspired Beauchamp and co-workers to investigate the reactions of protonated benzene and its substituted derivatives with neutral D2O in an ion cyclotron mass spectrometer [2]. Interestingly, the eficacy of the H/D exchange reaction was found to be dependent on both the protonation of the aromatic ring and the identity of any aromatic substituents, because none of the compounds containing electron-withdrawing groups were observed to undergo exchange. Furthermore, the kinetics and extents of the reaction were shown to differ for structural isomers such as o- and m-diluorobenzenes. At irst glance, the results appeared to be contradictory to those reported previously by Hunt [1] in that benzene and other aromatic molecules were observed to form isotope exchange products. However, the key difference between the two experiments was determined to be, not surprisingly, the identities of the pair of reactants in each case; this critical distinction explained the variation in results observed. Ultimately, Hunt and co-workers continued their research into isotope exchange under CI conditions by exploring the differences in exchange between various deuterated reagent gases (D2O, C2H5OD, and ND3) [3]. Dramatic differences in the CI mass spectra were observed as the deuterating agent/CI reagent gas was varied. In some cases, analytes that were observed previously to exchange very little with a particular deuterating agent were observed to exchange all of the available hydrogen atoms with alternate agents. In contrast, other ion/CI-reagent pairings produced no additional exchange regardless of the deuterating agent/CI reagent gas used. Furthermore, in addition to positive-mode CI using protonated species, CI in the negative ion mode was investigated also and similar isotope exchange reactions were observed. The importance of this investigation became clear during the interpretation of the experimental results. Foremost, the eficiency of the exchange reaction could be ascribed to the relative difference in basicities or acidities of the reagent and analyte, since the extent of deuterium incorporation was shown to decrease as the proton afinity difference between the reactant and analyte increased. Furthermore, Hunt and co-workers were the irst to propose formally the formation of an ion–molecule complex as a necessary condition for H/D exchange; they argued that exchange must occur during the lifetime of this complex. Years of subsequent research have continued to afirm this hypothesis. Finally, the authors speculated that the observed isotopeexchange reaction was dependent on temperature, especially as it relates to internal energy of the ions investigated. Collectively, these concepts became the foundation for continuing research into the mechanism of the gas-phase H/D exchange reaction and spurred many other groups to investigate the utility of the gas-phase H/D exchange reaction as an analytical tool.

2.1.2

THEORY OF GAS-PHASE HYDROGEN/DEUTERIUM (H/D) EXCHANGE EXPERIMENTS

Gas-phase H/D exchange reactions constitute a speciic type of ion/molecule reaction. The vast majority of these reactions involve gas-phase ions containing acidic hydrogen atoms bound to oxygen, nitrogen, or sulfur atoms (that is, acidic hydrogen atoms in alcohols, phenols, carboxylic acids, amines, amides, or thiols). However, speciic cases of H/D exchange of non-labile hydrogen atoms (that is, those bound to atoms such as

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carbon) have been reported also [4–6]. In these instances, subtle nuances in the local electronic environment of the carbon atom produced unusually acidic hydrogen atoms that were amenable to exchange. Again, unlike CI, the ion participant in the ion/molecule reaction in gas-phase H/D exchange is the analyte under study while the neutral molecule is one of many deuterating agents. 2.1.2.1 Deuterating Agents Popular choices for deuterating agents for gas-phase H/D exchange include D2O, CH3OD, CD3OD, ND3, DI, and D2S, although more exotic reagents such as CF3CH2OD, C6H5CH2OD, and C6D5OD have been used also. Historically, the choice of deuterating agent has been based primarily on its gas-phase acidity or basicity relative to that of the analyte of interest, although practical factors such as the vapor pressure of the deuterating agent and corrosivity of the agent toward instrumentation components may inluence the decision also. In the gas phase, the acidity of a molecule is deined as the molar Gibbs energy, ΔG, required to dissociate it heterolytically into a proton and an anion [7]. H/D exchange reactions involve both analyte ions and deuterating agents that contain relatively acidic hydrogen or deuterium atoms. Likewise, the gas-phase basicity is the Gibbs energy of the associated protonation reaction, and the H/D exchange reaction involves relatively basic oxygen, nitrogen, or sulfur atoms that accept the exchanged deuteron. Table 2.1 lists the gas-phase acidities and basicities of several common deuterating agents. 2.1.2.2 Proposed Mechanisms Gas-phase H/D exchange is a complicated process that may depend on a number of factors including the relative acidity and basicity of the analyte and deuterating agent, the proximity of the charge site to the potential exchange site, the intramolecular interaction of the labile hydrogen atoms with other basic sites, the internal energy of the analyte ion, and the frequency of collisions and energetics of ion–molecule complexation. Therefore, different reaction conditions (for example, different deuterating agents, reagent pressures, ion temperatures) can promote different levels of H/D

TABLE 2.1 Gas-Phase Acidity and Basicity of Common Deuterating Agents Deuterating Agent Hydroiodic acid (DI) Deuterium sulide (D2S) Deuterated ethanol (C2D5OD) Deuterated methanol (CD3OD) Deuterium oxide (D2O) Deuterated ammonia (ND3)

Gas-Phase Acidity (kJ mol −1)

Gas-Phase Basicity (kJ mol −1)

1315 1469 1561 1598 1636 1690

601 674 746 725 660 819

Source: All data are taken from Hunter, E.P.; Lias, S.G. NIST Chemistry WebBook. National Institute of Standards and Technology: Gaithersburg, MD, 1998.

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Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

exchange. Furthermore, different mechanisms are favored depending on the reactivity and gas-phase conformations of the reactants. The most fundamental principle of gas-phase H/D exchange involves the difference in basicity (or acidity) of the analyte and deuterating agent. In general, H/D exchange is favored when either the gas-phase acidity of the analyte and deuterating agent or gas-phase basicity of the analyte and deuterating agent are similar (that is, differing by less than 84 kJ mol−1). In these cases, the proton and deuteron transfer can occur via a relatively thermoneutral reaction. Indeed, this general statement has held true for many small molecule H/D exchange studies [7,9], but not for others involving amino acids and peptides, where exchange has been shown to occur when the difference in gas-phase basicities is greater than 84 kJ mol−1 [10]. Ultimately, the difference in gas-phase acidity (or basicity) of the reaction participants is only one key aspect of the H/D exchange process. Another factor, which has a major impact on gas-phase H/D exchange, is the proximity of the charge site to the labile hydrogen atoms. In some cases, the mechanism involved in the H/D exchange requires the two reactive sites to be relatively close in order to facilitate ion–molecule complexation and subsequent exchange. The relay mechanism shown in Figure 2.1 is an important example. This mechanism requires the formation of a stable ion–molecule complex in which a deuterating reagent bridges the gap between a charge site and a labile hydrogen atom, thereby allowing indirect interaction of the two remote sites. In this case, the charge site serves as the deuterium acceptor in the exchange process, while the labile hydrogen is transferred to the deuterating agent.

O

O H

D

O

H

D

O

O

D

O

P

HO

O

Nucleobase

O

O

P O

O

D

O

Nucleobase

O

H

O

O

D

O

O

P

HO

O

HO

Nucleobase

O

O

P

HO

O

Nucleobase

O

D

D

O H O D

FIGURE 2.1 H/D exchange via the relay mechanism. The reaction is shown for a generic mononucleotide anion reacting with D2O. (Reproduced from Chipuk, J.E.; Brodbelt, J.S. J. Am. Soc. Mass Spectrom. 2007, 18, 724–736. With permission from Elsevier.)

40

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V M+H or M–H O

R

O

D

C

O O

H

D

M+H or M–H

M+H or M–H

R

-

D

C

O O

H

D

R

O

D

O

H

C

O

D

FIGURE 2.2 H/D exchange via a Flip-Flop mechanism. The reaction is shown for a generic carboxylic acid reacting with deuterium oxide (D2O).

In contrast, the lip-lop mechanism shown in Figure 2.2 is independent of the charge site and involves the interaction of the deuterating reagent directly with the labile hydrogen atom. For this route, the ion–molecule complex forms a pseudo-ring structure by the attraction of partially-charged participants, and exchange occurs as one of the potential outcomes of its disassembly. In many cases, the inherent gas-phase acidity or basicity of a deuterating agent results in the reagent favoring particular exchange mechanisms regardless of the ion involved. D2O is one example, as the relatively low acidity and basicity result in D2O reacting typically via the relay mechanism shown in Figure 2.1. Another example is deuterated ammonia, ND3. In this case, the basicity of the deuterating agent favors the abstraction of a proton from positively-charged ions, such as the N-terminus of peptides, in what would be a nominally-endothermic process. This transition results in the formation of an ammonium ion [ND3H] + and is typically accompanied by simultaneous solvation of the resultant ion. Subsequent shuttling of protons via tautomerism or other resonance may then result in conditions favorable for the transfer of one of the deuterium atoms to the analyte, resulting effectively in H/D exchange. The gas-phase chemistry of ND3 is well suited to this onium mechanism, making reactions of ND3 with positive ions among the most eficient. In contrast, the low gas-phase basicity of D2O alone would be likely to preclude this type of exchange. While the aforementioned exchange mechanisms have received the most attention, the debate over the mechanism of gas-phase H/D exchange reactions continues. While many groups have concluded that speciic results seem to be correlated to a particular mechanism, it is likely that in many other cases multiple mechanisms are contributing simultaneously.

2.2

PRACTICAL ASPECTS OF GAS-PHASE HYDROGEN/ DEUTERIUM (H/D) EXCHANGE

Gas-phase H/D experiments are carried out in a number of ways and utilize a variety of instrumentation conigurations. In many instances, the results of the experiment are determined by the conditions utilized, and particular attention must be paid to the methodology employed. Furthermore, interpretation of the exchange data often requires additional insight when compared to typical mass spectral analysis, and rigorous molecular modeling is often a companion effort. Collectively, these complexities necessitate extra discussion of the process of conducting gas-phase H/D exchange experiments in ion-trapping instruments.

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

2.2.1

41

MOTIVATION FOR GAS-PHASE HYDROGEN/DEUTERIUM (H/D) EXCHANGE EXPERIMENTS

Gas-phase H/D exchange experiments are carried out typically with one of three major goals in mind: to elucidate the gas-phase structure of a particular ion; to differentiate between structurally-isomeric ions; or to assess the reactivity of ions in the absence of solvent. Many of these experiments are complemented by extensive computational modeling of the ion and/or ion–molecule complex as a means of validating the experimental results and, thereby, furthering the understanding of the mechanism of the experiment. The search for structural information of ions in the gas phase is relevant for all ions but, in recent years, the search has been targeted especially at large biomolecules where secondary structures are complex and critical for function in solution. For example, these experiments may be aimed at elucidating the folding and unfolding of proteins or probing the tertiary structure of oligonucleotides such as DNA quadruplexes. In doing so, these results are often complementary to those obtained by other experimental techniques such as ion-mobility measurements. Ultimately, the motivation for all of these experiments is to understand the readily accessible gas-phase conformations, how they interconvert, and how they may be correlated with structures and behaviors of molecules in the native solution state. In the case of isomeric differentiation, gas-phase H/D exchange serves to complement other techniques such collisionally-induced dissociation (CID), infrared multi-photon dissociation (IRMPD), or ultra-violet photon dissociation (UVPD). For small molecules, these dissociation techniques may at times prove to be inadequate to differentiate isomeric or enantiomeric species, and thus gas-phase H/D exchange may be an alternate way to determine structural differences, provided that the isomers contain exchangeable acidic hydrogen atoms. For oligomeric species such as peptides and oligonucleotides, differences in H/D exchange patterns may assist in determining the sequence of amino acids or nucleotides. While gasphase H/D exchange is not likely to replace more powerful bottom-up or top-down sequencing techniques, it can be used in speciic cases as a secondary means of qualitative identiication. Gas-phase H/D exchange is also useful for chemists interested in probing fundamental aspects of ion reactivity and thermochemistry. Reactions involving neutral deuterated molecules of varying acidity allow an intricate method for assessing the tendency of ions to react via gas-phase acid–base reactions. In some cases, these fundamental experiments produce results that are contrary to what is believed to take place in solution, again highlighting the unique chemistry that can take place in the absence of a ubiquitous solvent. In addition, reactions can be performed, on occasion, in relatively-controlled conditions where both the kinetic and potential energies of the ions can be calculated and, in some cases, manipulated via additional blackbody heating, off-resonance excitation, or direct activation via interaction with electromagnetic radiation, such as with an IR laser. Ultimately, these types of experiments are performed to increase the fundamental knowledge base of gas-phase ion chemistry.

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

2.2.2 INSTRUMENTATION FOR GAS-PHASE HYDROGEN/DEUTERIUM (H/D) EXCHANGE EXPERIMENTS In the gas-phase H/D exchange process, the lifetime of the ion–molecule complex is critical, as results from numerous experiments have demonstrated that reactions proceed at varying rates. It is, therefore, not surprising that the bulk of the gas-phase H/D exchange studies have been performed using either a quadrupole ion trap or a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, because these two techniques are so well suited to trapping ions for variable periods to allow kinetic analysis. While the two methodologies have many fundamental similarities, they also have several inherent differences, including the temperatures of ions, the number of collisions that ions undergo with the reagent or buffer gas (due to the dramatic difference in operating pressures), and the total reaction time. These factors may inluence the results of the exchange reactions. The following sections will focus primarily on conducting H/D exchange reactions in each of two-dimensional and three-dimensional quadrupole-ion traps. 2.2.2.1 Ion Trapping for Gas-Phase Hydrogen/Deuterium (H/D) Exchange While the majority of H/D exchange reactions reported in the literature have been conducted in FT-ICR mass spectrometers, numerous studies have been reported that utilize quadrupole-ion traps. Amongst these, the vast majority have utilized threedimensional hyperbolic quadrupoles, such as Thermo Finnigan LCQ [11–14] or Hitachi 3DQ mass spectrometer [5,6,15–17]. The less frequent use of two-dimensional ion traps, such as a linear ion trap (LIT) [18–21], is likely due to the simple fact that there are fewer research groups that possess the instrumentation and have adapted it for H/D exchange studies. Other ion-trap conigurations, such as a cylindrical ion trap (CIT) or rectilinear ion trap (RIT), should also be amenable to conducting H/D-exchange reactions, since these instruments have been reported to be useful for conducting other ion/molecule reactions [22]. In theory, all ion traps with suficient resolution should be amenable to conducting gas-phase H/D exchange reactions, provided that they can be integrated with an appropriate ion source and maintain proper operating pressure when integrated with a deuterating reagent inlet system. Ion introduction for gas-phase H/D exchange is performed typically at atmospheric pressure, and most often through the use of ESI. However, some examples that utilize Matrix-assisted laser desorption/ionization (MALDI) [23] can be found in the literature and gas-phase isotope exchange studies also utilize CI [1,24]. ESI lends itself particularly well to gas-phase H/D exchange experiments because protonation and deprotonation often involve relatively basic groups, such as amines, or relatively acidic hydrogen atoms such as those found in phosphates. In many cases, these sites either contain or are in close proximity to more than one labile hydrogen atom (for example, terminal phosphates in oligonucleotides). 2.2.2.2 Reagent Inlet Systems Undertaking H/D exchange reactions in an ion trap mass spectrometer requires a means of introducing a deuterating agent. Several popular methods include introduction of deuterating agent through the helium bath gas line [11–14], through a leak

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

43

valve directly to the ion trap or to the ion trap vacuum manifold [5,6,15–17], via direct and continuous infusion to the ion trap chamber [18–21], or in some cases through a pulsed-valve system [23]. Introduction of neutral reagent through the helium bath gas line of a Thermo Finnigan LCQ was described irst by Gronert [25]. In this system, a measured low of liquid deuterating agent is added via a syringe pump to a measured low of helium. The rapid vaporization that takes place at the syringe tip allows for a range of mixing ratios of the reagent and the helium gas. While most of the total low, including the deuterating agent, is directed to a low meter, a small portion is drawn into the ion trap. In many cases, the internal down-stepping regulation of helium is bypassed and helium is, instead, delivered directly to the trap at reduced pressures. This adjustment reduces the dead volume of the introduction pathway and speeds up the response of the system to changes in the low rate, and hence concentration, of the deuterating agent. This type of reagent inlet system allows for measurable and inely tunable deuterating reagent pressures into the ion trap with an estimated uncertainty of ca 20%, most of which is caused by the uncertainty in measuring the helium pressure itself [25]. The primary beneit is the ability to add small amounts of deuterating agent in a reproducible manner, which leads ultimately to the ability to conduct kinetic reaction measurements at a series of known reagent concentrations. The primary downside of this approach is contamination of the helium bath gas line with the deuterating reagent. While this contamination may be of lesser concern when the instrumentation is utilized exclusively for ion/molecule reactions such as H/D exchange, normal operation of the instrument under standard analytical conditions may be prone to interferences caused by the presence of reactive contaminants. Introduction of the neutral deuterating reagent through a leak valve is also an effective way to implement gas-phase H/D exchange [5,6,14–17]. In this approach, a liquid or gaseous reagent stored in a secondary vessel (for example, a glass inger tube for liquids or a lecture bottle for gases) is subjected to the vacuum of the mass spectrometer through an adjustable needle valve. When the reagent is a volatile or moderatelyvolatile liquid at room temperature, such as D2O or CD3OD, the reduced pressure will increase the vapor pressure of the liquid, and gas-phase neutral deuterating agent will be transferred to the ion trap via the pressure differential. Naturally, gaseous reagents such as D2S and ND3 are transferred easily in a similar manner. In some cases, the deuterating agent is leaked directly into the interior volume of the ion trap via a gas line plumbed through one of the hyperbolic end-cap electrodes of the trap. In other cases, the reagent is leaked into the vacuum manifold where it ills the evacuated space and, eventually, equilibrates with the helium bath gas. Introduction of deuterating agent via a leak valve has the advantages of maintaining the integrity of the helium transfer line and providing easily suficient reactant to ensure that the concentration of the deuterating reagent is not the limiting factor in the exchange reaction. The primary disadvantage of this introduction method, relative to the helium gas line method, is the inability to control precisely the addition of small amounts of deuterating agent. In these instances, there is nearly always a stoichiometric excess of the deuterated reactant and exchange reactions that are concentration dependent and have very fast exchange rates become dificult to model kinetically. Therefore, the leak valve method may favor comparison of the extent of H/D exchange among different species over rigorous modeling of the reaction kinetics for rapidly exchanging systems.

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

A method for direct infusion of a deuterating agent to a RIT has been described also [18–21]. This procedure is similar to the leak valve approach except that a 0.9 mm nozzle is placed 4 mm radially from the center of the ion trap and is used to deliver the deuterating agent directly to the interior of the ion trap via the formation of a free jet. While H/D exchange is believed to occur in the free-jet expansion, it is assumed to be more extensive in the surrounding trapping environment as continuous infusion builds up a background pressure of deuterating agent. Calculations supporting this assumption suggest that there is a greater number density of deuterating agent molecules in the background compared to the free-jet area. In addition to these continuous introduction schemes, instruments have been constructed that utilize a pulsed-valve method for introducing the deuterating agent [23]. While this method is similar to the leak valve method, it offers the advantage of allowing the ion trap to ‘reset’ after each mass analysis scan and, therefore, to allow accumulation of ions within the ion trap before they are subjected to the neutral deuterating agent. In this way, all ions have a similar opportunity for exchange regardless of whether they were ionized and accumulated at the beginning of the ionization period or toward the end. The primary disadvantage of this method is that high-pressure pulses reduce the reaction stability produced by equilibrating the ion trap to the deuterating agent. The inherent variability in the pulsing system may change signiicantly the concentration of the deuterating agent on a scan-to-scan basis. In so doing, the variability of the exchange results may increase, especially as concentrations are reduced and when the kinetics of rapidly-exchanging systems are being investigated.

2.2.3

METHODS

As mentioned previously, gas-phase H/D exchange reactions are a form of ion/molecule reaction in which an analyte ion reacts with a neutral deuterated molecule within the conines of an ion-trap or ion-drift tube. The product of the reaction is a covalent exchange of hydrogen for deuterium in what amounts to a double replacement reaction that is expected to proceed through a stable ion–molecule complex. Like other organic reactions, the extent of gas-phase H/D exchange reactions depends on the number density of molecules of each reactant species present and the time during which they are allowed to react. 2.2.3.1 Typical Reaction Conditions The pressure of the ion-trapping system is critical to the rate, and, ultimately, to the extent, of any gas-phase H/D exchange reaction. Deuterating agents are introduced typically to the trap at pressures between 0.1 and 3.0 mTorr with higher pressures favoring more rapid exchange but, potentially, compromising mass spectral resolution. These pressures are approximately four orders of magnitude larger than those used in an FT-ICR cell and thus, have led several researchers to conduct H/D exchange experiments in parallel on both types of instruments [13,14,26]. Reaction times typically range from milliseconds to 10 s, although exchange times up to 40 s have been accomplished [13]. Furthermore, when a pulsed deuterating agent introduction system is not used, the actual reaction time is approximately the sum of the ion accumulation time, any isolation time, and any exchange time, since the deuterating

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

45

agent is present within the ion trap throughout the entire analysis. Exchange times of less than 10 s are far shorter than those used typically in FT-ICR studies. Therefore, the two mass analyzers provide somewhat complementary information in that a quadrupole ion trap can monitor the reaction of high concentrations of both ions and deuterating agents for a relatively short period of time, while FT-ICR mass analyzers monitor typically much smaller reactant concentrations but for much longer time periods. Aside from the reagent pressure and reaction times, H/D exchange experiments are sensitive to contamination from hydrogen sources such as water or methanol. Because these ions provide a means of exchange of deuterated species back to unexchanged ones, a conditioning procedure is carried out normally, whereby the ion trap is exposed to the gas-phase deuterating agent for up to one hour prior to analysis, so as to remove any unwanted contamination in the ion trap. As with other analyses, it is often the case that samples are analyzed multiple times across different days to assess reproducibility. This practice is especially important when making comparisons between reaction rates of isomeric species, because many reactions are very sensitive to the deuterating reagent pressure and this pressure may be dificult to control on a day-to-day basis. Therefore, whenever possible, series of analyses are run consecutively on a given day to control against pressure variation. 2.2.3.2 Mass Spectral Interpretation The evolution of a typical gas-phase H/D exchange reaction can be monitored by varying the reaction time while keeping constant the accumulation time, isolation time, and pressure of the deuterating agent. An example of such an evolution is shown in Figure 2.3. As the reaction time increases, the relative intensity of the isolated analyte ion, m/z 306, decreases as labile hydrogen atoms are exchanged for deuterium atoms. The relative intensities of m/z 307, 308, and 309 increase initially and then decrease, while that of m/z 310 increases until, after 10 s, the exchange of all four labile hydrogen atoms by deuterium atoms is complete. The ion of m/z 310 in Figure 2.3(d) has four deuterium atoms and represents the complete H/D exchange. The ion of m/z 311 does not represent additional exchange because it is the 13C isotopic analog of the peak of m/z 310. Indeed, contributions to a particular ion population m/z from non-exchanged isotopes, such as 13C, need to be corrected prior to performing any detailed analysis of the relative peak intensities. 2.2.3.3 Reaction Kinetics Many gas-phase H/D exchange experiments are performed to assess not only the extent to which the reaction takes place, but also the rate at which the reaction proceeds. Besides providing valuable insight into gas-phase ion reactivity, reaction rates are investigated because they often provide additional information that can be used to differentiate isomeric species or to give indications about the secondary structure of larger biomolecules in the gas phase. To perform a kinetic analysis, a series of experiments is performed at various exchange times ranging over several orders of magnitude (for example, 0 ms to 10 s). After correcting the peak intensities for non-exchanged isotopic contributions such as 13C, the series of data is it typically to a system of coupled ordinary differential equations using programs such as KinFit [27] to determine the rate constants.

46

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (b)

(a)

15,000

25,000

D0 15,000

*306

10,000

Intensity

Intensity

20,000

D2

10,000 *306 5000

5000 0 304 305 306 307 308 309 310 311 312 313 314 m/z

0 304 305 306 307 308 309 310 311 312 313 314 m/z

(d)

(c)

30,000

10,000

5000 *306

D4

25,000 Intensity

Intensity

D3

20,000 15,000 10,000

*306

5000 0 304 305 306 307 308 309 310 311 312 313 314 m/z

0 304 305 306 307 308 309 310 311 312 313 314 m/z

FIGURE 2.3 Representative mass spectra for the reaction of deprotonated 2-deoxy-5-cytidine monophosphate (5-dCMP) with D2O in a quadrupole-ion trap. Reaction times are (a) 0 ms, (b) 250 ms, (c) 2000 ms, and (d) 10,000 ms. Peaks are labeled as Dn, where n equals the number of incorporated deuterium atoms. (Reproduced from Chipuk, J.E.; Brodbelt, J.S. J. Am. Soc. Mass Spectrom. 2007, 18, 724–736. With permission from Elsevier.)

A plot of the calculated rate constant equation for each exchange along with the exchange data is prepared usually as an illustrative tool. An example of such a plot is shown in Figure 2.4.

2.3

CURRENT AREAS OF RESEARCH

Initially, the most common application of H/D exchange was aimed at identiication of the presence of particular acidic or basic sites in a molecule. This concept evolved quickly into investigations to distinguish between speciic structural and stereoisomers of trapped ions. The invention of soft ionization techniques, such as MALDI and especially ESI, led to a completely different set of applications for gas-phase H/D exchange. The rapidity with which these new ionization techniques were introduced allowed the study of a wide range of biomolecules such as peptides, proteins, and oligonucleotides in the gas phase, and used the internal volume of the ion trap as a conigurable chemical reactor. While some of the research in this area has utilized H/D exchange to differentiate between structural variations (that is, sequence of subunits), the majority of

47

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps 1 OH

0.8

OH

Relative abundance

OCH3

CH2OH

CH3OH O OH OH

D(0) 0.6

O

O

O

O OH

O

OH

0.4 D(1) 0.2

D(2) D(3)

D(4)

0 0

2

4

6

8

10

H/D exchange time (s)

FIGURE 2.4 Kinetic plot of the H/D exchange reactions of the deprotonated lavonoid, neohesperidin, itted with KinFit. D(0) represents the initial precursor ion, and D(1), D(2), D(3), and D(4) represent ions incorporating from one to four deuterium atoms, respectively. (Reproduced from Zhang, J.; Brodbelt, J.S. J. Am. Chem. Soc. 2004, 126, 5906–5919. With permission from American Chemical Society.)

the work aims to investigate a much broader question, that of the conformation and secondary structures of these macromolecules in the gas phase.

2.3.1

SMALL MOLECULES

Research in the area of small molecule gas-phase H/D exchange in ion traps has focused primarily on two fronts: (1) model systems to investigate the mechanisms of H/D exchange; and (2) isomer differentiation. 2.3.1.1 Fundamental Studies of Model Compounds A classic example of a mechanism-motivated H/D study involved the exchange behavior of various polyamines that formed singly-protonated, doubly-protonated, and sodium- or potassium-cationized species upon ESI [28]. The goal was to determine the impact of various factors such as ligand lexibility, size, basicity, and location of basic groups on the H/D-exchange reaction. It was shown that, under identical conditions, exchange with singly-protonated analytes was faster and more eficient than exchange with either sodium- or potassium-cationized complexes. These results were explained by either the loss of a low energy exchange pathway between the alkali metal-cationized species and the reagent gas, or by a structural change that was induced by the presence of a bulkier cation as compared to a proton. Furthermore, the H/D exchange of the doubly-protonated species was found to be much more eficient than for the singly-protonated species. Ultimately, it was concluded that gas-phase

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (b) 50,000 OH D(0) O 45,000 O 40,000 H 35,000 OH 30,000 25,000 H H 20,000 H 15,000 D(1) + 13C D(0) PA 10,000 5000 0 160 161 162 163 164 165 166 167 168 169 170 Mass (m/z)

D(2) 50,000 O OH 45,000 40,000 H 35,000 H 30,000 25,000 O 20,000 H 15,000 H OH D(0) 10,000 13 CD(2) D(1) IPA 5000 0 160 161 162 163 164 165 166 167 168 169 170 Mass (m/z)

(d)

(c)

Intensity

Intensity

Intensity

(a)

O D(1) + 13C D(0) OH 16,000 14,000 H D(0) 12,000 H 10,000 8000 H H 6000 OH O 13 4000 C D(1) TPA 2000 0 160 161 162 163 164 165 166 167 168 169 170 Mass (m/z)

Intensity

48

O OH 18,000 D(0) 16,000 H H 14,000 H 12,000 H 10,000 8000 D(1) + 13C D(0) H H 6000 4000 OH O 13 C D(1) 2000 NAPA 0 210 211 212 213 214 215 216 217 218 219 220 Mass (m/z)

FIGURE 2.5 H/D exchange mass spectra for deprotonated (a) phthalic acid, (b) isophthalic acid, (c) terephthalic acid, and (d) 2,6-naphthalic acid after 10 s exchange with D2O. (Reproduced from Chipuk, J.E.; Brodbelt, J.S. Int. J. Mass Spectrom. 2007, 267, 98–108. With permission from Elsevier.)

basicity, conformation of the ion, and interaction of the ion with the exchange reagent all play a role in the H/D exchange behavior. Another example of this type of study involved the H/D exchange reactions of deprotonated aromatic dicarboxylic acids [5]. In this case, the intent was to determine the difference in observed H/D exchange behavior as the relative position of the two carboxylic acid groups varied. Figure 2.5 summarizes the H/D exchange behavior and illustrates that the spatial relationship of the deprotonated sites has a tremendous impact on the extent of the H/D exchange. The exchange mass spectrum in Figure 2.5a illustrates the inluence of intramolecular hydrogen bonding between the deprotonated site and proximate labile hydrogen atoms and how it may decrease the proclivity of these hydrogen atoms to exchange. The mass spectra in Figure 2.5c and d describe the opposite scenario, and illustrate that H/D exchange may occur via mechanisms that do not require the proximity of the labile hydrogen atoms to the deprotonated site, albeit at a much slower rate. Finally, the mass spectrum in Figure 2.5b shows the H/D exchange of traditionally non-labile hydrogen atoms. In this instance, the aromatic hydrogen located between the two carboxylic acid groups is unusually acidic and may allow the formation of carbanions that react subsequently with excess deuterating agent. While the differentiation between various phthalic acid isomers is not a chemistry landmark analytical application, the impact of the results extends beyond the differentiation of isomers and illustrates the intricacies of the gas-phase H/D exchange process. The conclusions reinforced previous studies that suggested that H/D exchange

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

49

behavior was dependent on gas-phase acidity, intramolecular hydrogen bonding, and intermolecular hydrogen bonding in the ion–molecule complex. In addition, it was suggested that multiple reaction mechanisms could occur. 2.3.1.2 Isomer Differentiation The utility of gas-phase H/D exchange as a method of isomer differentiation is exempliied by an in-depth study of the H/D exchange behavior of ive isomeric lavonoid glycosides and their corresponding aglycons [15]. Although most lavonoids have a common phenyl-benzopyrone skeleton, they differ with respect to each other by the positions of hydroxyl and methoxy groups; the location, number, and identities of saccharides involved in glycosylation; and the intersaccharide linkage in the case of polysaccharides [15]. Figure 2.6 shows several H/D-exchange mass spectra obtained for ive isomeric lavonoid monosaccharides of m/z 447. In Figure 2.6a and d, H/D exchange was very minimal, while in Figure 2.6c and e, the exchange was quite extensive. Obviously, the relative position and location of the labile hydrogen atoms was important to the exchange behavior. Moreover, unlike other molecules where a particular site is favored for protonation or deprotonation, the lavonoids contain many seemingly equivalent sites for deprotonation during ESI. Through extensive experimentation and high-level computational calculations, it was concluded that the location of the most likely deprotonation site (that is, the most acidic hydrogen), the ability of the charge to migrate via resonance, the relative acidities of the various labile hydrogen and deuterium atoms, and the lexibility of the various moieties to adopt favorable gas-phase conformations all played a role in determining the H/D exchange behavior. It was, therefore, a summation of all of these factors that resulted in the H/D exchange being remarkably sensitive to the subtle structural differences within the lavonoid isomers. Another interesting example of isomer differentiation by gas-phase H/D exchange was reported for catechins [6]. In this case the low-energy nature of the H/D exchange process was utilized to explore the possibility of differentiating various stereoisomers of both galloylated and non-galloylated species. Interestingly, stereoisomerism was found to have little effect on the reaction kinetics of the non-galloylated catechins with deuterium oxide, while the galloylated species, which differed only in the chirality of one the carbon atoms on the lavonoid ring, had distinctive H/D exchange kinetics. Furthermore, all of the non-galloylated catechin isomers were observed to exchange aromatic or allylic non-labile hydrogen atoms. An accompanying computational investigation showed that the addition of the gallate moiety increased signiicantly the gas-phase acidity of the hydroxyl hydrogen atoms and, thus, disfavored dramatically the scrambling of these hydrogen atoms to non-labile aromatic sites; in contrast, the non-galloylated species were shown to be prone to this type of rearrangement. The differentiation of isomers by gas-phase H/D exchange continues to be explored. The most compelling experiments are aimed at distinguishing quickly and accurately compounds that either cannot be distinguished by higher energy dissociation methods, such as CID, or require lengthy chromatographic runs, as in liquid chromatographymass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS) studies. These isomer differentiation studies tend to reveal information about the effects of gas-phase conformations and thermochemistry that can be used to understand the reactions of more complex molecules that contain similar functional groups.

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

(a)

Luteolin-4´-O-glucoside

(b)

CH2CH

*447

OH OH O

Intensity

Intensity

OH

O

O

O

OH OH

*447

OH OH

O

O

452

456

444

460

448

452

456

460

Mass (m/z)

Mass (m/z) (c)

OH

CH2OH

O

HO

448

OH

OH OH

444

Luteolin-7-O-glucoside

O

(d)

Orientin

Astragalin OH

CH2OH O

OH

OH

HO

OH OH

O

*447 OH

444

448

O

HO

452 456 Mass (m/z)

O

Intensity

Intensity

OH

*447

OH

O CH2OH O OH

O

OH

444

460

(e)

448

Quercitrin

452 456 Mass (m/z)

OH

460

OH OH O

Intensity

HO

O OH

O CH3

O OH OH

*447 OH

444

448

452 456 Mass (m/z)

460

FIGURE 2.6 H/D exchange of ive deprotonated lavonoid monosaccharides (a) Luteolin4′-O-glucoside, (b) Luteolin-7-O-glucoside, (c) Orientin, (d) Astragalin, and (e) Quercitrin after 10 s reaction with D2O. (Reproduced from Zhang, J.; Brodbelt, J.S. J. Am. Chem. Soc. 2004, 126, 5906–5919. With permission from American Chemical Society.)

2.3.2

PEPTIDES AND PROTEINS

Investigating the behavior of biomolecules such as peptides and proteins is one of the most important and challenging aspects of modern analytical chemistry. While solution-phase studies offer the advantage of being similar to the native biological environment, these experiments are often time consuming and sensitive to variations in the solution conditions. In contrast, gas-phase studies are more eficient and can

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

51

be performed in the absence of solvent interferences. Indeed, proteomics has become a commercialized endeavor and the sequencing of peptides and proteins via mass spectrometric techniques has become standard practice in many laboratories. While the sequence of a peptide or protein is valuable information, the secondary and tertiary structures of the biomolecules are known to be critical to their biological activity. Therefore, additional experiments such as gas-phase H/D exchange have been designed to investigate the higher-order structures of these biomolecular ions. Ultimately, these gas-phase studies are aimed at providing insight into the behavior of the biomolecules in their native, solution-phase environment. 2.3.2.1 Model Peptides Numerous H/D exchange studies of peptides have been reported. Initial work on glycine oligomers using a 3D quadrupole ion trap conirmed not only the previous indings obtained by FT-ICR mass spectrometry but included studies of the collision-induced product ions as well as methylated oligomers [11]. Studies of H/D exchange reactions have been conducted also in concert with ion mobility studies and high-level computations to probe the gas-phase H/D exchange mechanisms active in a family of singly-protonated pentapeptides [26]. While a salt bridge mechanism appeared initially to be responsible for the H/D exchange, the authors concluded that, in fact, this structure was only a kinetic intermediate and that exchange followed from a relay mechanism. Peptides often contain highly basic residues and these oligomers provide an exceptional opportunity for the study of the impact of multiple charge sites on the gas-phase structure. Bradykinin is one of the polypeptides studied most often in mass spectrometry, due primarily to its manageable mass (1060.21 g mol−1) and two highly basic arginine residues. Gas-phase H/D exchange reactions of bradykinin have been studied using a MALDI-QIT instrument [19], an ESI-QIT instrument [14,29–30], and an ESI-LIT-TOF instrument [19]. Figure 2.7 shows a series of H/D exchange mass spectra for bradykinin ions collected using CD3OD as a reagent gas in an LIT-TOF mass spectrometer. Among the most signiicant indings is the evidence that the triply-protonated species (that is, [M + 3H]3 + ) and the doubly-protonated species (that is, [M + 2H]2 + ) of bradykinin produced multiple and distinct gas-phase conformations, as indicated by the corresponding bi-modal H/D exchange mass spectra [19,29]. The dissociation of these ‘fast’ and ‘slow’ exchanging species was studied subsequently in both FT-ICR and quadrupole ion trap instruments [14]. Another important inding was that the H/D exchange proceeded through a relay mechanism when D2O or DI was used as the deuterating agent, but probably through an alternate mechanism when ND3 was used as the deuterating agent [19,29–30]. Furthermore, results for bradykinin revealed that multiply-charged species exchanged faster than did singly-charged species and exchange of sodium-cationized species was very limited. 2.3.2.2 Proteins Gas-phase H/D exchange of proteins in ion-trapping instruments is a very challenging experiment. One noticeable difference between these experiments and those conducted on smaller molecules is the resolution of labeled species that can be attained typically. This difference stems directly from the fundamental operating

52

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a) BK+2H+

(g) BK+H+ 0s

(b)

(h) 0s

(c)

(i) 1s

(d)

(j) 5s

(e)

(k) 20s

(f )

(l) 80s

530 532 534 536

538 540 1060 m/z

1065

1070

1075

FIGURE 2.7 Mass spectra of doubly (a–f) and singly (g–l) protonated bradykinin molecules with different storage times, listed to the right of the igure. (a) and (g) were recorded with 7.4 × 10 –3 Torr of nitrogen in the LIT chamber while all others were recorded with a 1.7 × 10 –3 Torr base pressure of nitrogen and 5.7 × 10 –3 Torr of CD3OD in the trap chamber. (Reproduced from Mao, D.; Douglas, D.J. J. Am. Soc. Mass Spectrom. 2003, 14, 85–94. With permission from Elsevier.)

considerations of ESI and the limitations of ion-trapping instruments. Unlike small molecule exchange, where ionization involves typically a single deprotonation or protonation reaction, ESI of proteins produces multiply-protonated and multiply-deprotonated species having much higher charge states. This phenomenon is extremely beneicial in that it extends effectively the mass range of the ion trap and allows species with high molecular weights to be investigated in the ion trap. Unfortunately, the shift in charge state is not accompanied by an increase in resolving power of the mass spectrometer. For example, H/D exchange of a singly-protonated molecule of m/z 1000 requires only that the mass analyzer be capable of differentiating between

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps

53

ions of m/z 1000 and m/z 1001 in order to monitor the isotopic exchange. In contrast, when a larger species with molecular weight of 10,000 Da is studied in the + 10 charge state, the mass spectrometer must be able to differentiate between ions of m/z 1001.0 and ions of m/z 1001.1 in order to detect a single isotopic exchange. Naturally, the gas-phase H/D-exchange analysis becomes more dificult as the mass and the most abundant charge state increase. Therefore, the utility of H/D exchange of proteins in ion-trapping instruments with moderate resolving power is limited to establishing trends in mass spectral shifts. Nonetheless, the technique provides unique insight into gas-phase conformations of proteins. For example, denatured proteins often undergo more extensive H/D exchange than do native proteins because the denatured proteins are less folded, thus, allowing access to interior active hydrogen atoms that were hydrogen bonded previously in the interior of the folded conformation. Indeed, the differentiation between folded and unfolded protein states is the primary focus of protein H/D exchange in both the gas phase and in solution. Studies of protein gas-phase H/D exchange in a LIT-TOF system include reactions of myoglobin and apomyoglobin with D2O and CD3OD [18]; lysozyme with D2O [20]; and ubiquitin, cytochrome c, apomyoglobin, and β-lactoglobulin with D2O [21]. An example of the H/D exchange of ubiquitin is shown in Figure 2.8. (a)

Relative intensity

(b)

(c)

(d)

(e)

1200

1220

1240

1260

m/z

FIGURE 2.8 Mass spectra of ubiquitin + 7 ions produced from 50:50 methanol:water at pH 2.0 at (a) and (b) 0, (c) 0.5 s, (d) 1 s, and (e) 5 s of trapping. The pressures in the trap chamber were (a) 7 mTorr N2 and (b)–(e) 2 mTorr N2 and 5 mTorr D2O. (Reproduced from Wright, P.J.; Zhang, J.; Douglas, D.J, J. Am. Soc. Mass Spectrom. 2008, 19, 1906–1913. With permission from Elsevier.)

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

Obviously, the resolution of the mass spectra is not suficient to discern the exact number of H/D exchanges. This particular series of mass spectra were used to correlate observations in the gas phase with similar ones obtained in solution, indicating that ubiquitin may have some memory of the solution conformation even after it is transferred to the gas phase via ESI. Similar results have been reported for studies utilizing a 3D quadrupole ion trap and CD3OD as the deuterating reagent [12].

2.3.3

NUCLEOSIDES, NUCLEOTIDES, AND OLIGONUCLEOTIDES

Fewer gas-phase H/D exchange experiments have been conducted on nucleic acids. The irst gas-phase H/D exchange study in a quadrupole ion trap involved the reaction of various nucleosides and nucleoside analogs with CH3OD and ND3 [24]. The impact of the gas-phase basicity of the deuterating agent on the observed exchange of singly protonated nucleosides was evaluated. Exchange mass spectra obtained in the positive ion mode conirmed that exchange with ND3 was more rapid and more extensive than with CH3OD; these results were rationalized based on the greater similarities in gas-phase basicities of the ND3 deuterating agent and nucleosides. However, it was reported also that variations in the position of the exchangeable hydrogen atoms relative to the suspected protonation site resulted in differences in exchange behavior, and that the presence of other non-participatory functional groups could impact upon the H/D exchange. A more recent study examined the H/D exchange of deoxyribose monophosphate nucleotides, the building blocks of oligonucleotides. However, unlike the nucleosides, the mononucleotides contain a highly acidic phosphate group, which can be deprotonated easily during ESI. The study was undertaken in the negative ion mode using a weakly acidic deuterating agent, D2O, that had been conirmed previously to promote exchange via a relay mechanism [16]. Variations in the exchange behaviors of the mononucleotide isomers conirmed that the reactions were dependent on both the identity of the nucleobase and the position of the phosphate moiety. It was concluded also that the distinction between mononucleotide isomers by H/D exchange pattern showed promise for distinguishing sequences of larger oligonucleotides. Energy-variation studies, conducted either by heating the ion trap or by activating the trapped ions with an IR laser, indicated that additional energy inluenced the H/D exchange pattern for the mononucleotides [16]. The investigation of the deoxyribose monophosphate nucleotides was followed by a subsequent investigation involving the simplest oligonucleotides, the dinucleotides. In this study, the H/D exchange reactions of dinucleotides containing only purine bases (that are, dAA, dAG, dGA, and dGG) and their 5′-monophosphate analogs (that are, 5′P-dAA, 5′P-dAG, 5′P-dGA, and 5′PdGG) were conducted using D2O in a quadrupole ion trap [17]. Signiicant differences in the rates and extents of exchange were found when the 5′-hydroxyl group of the dinucleotides was replaced by a phosphate functionality. Illustrative mass spectra are shown in Figure 2.9. Extensive and nucleobase-dependent exchange occurred for the deprotonated 5′-monophosphate dinucleotides, whereas the dinucleotides all exhibited essentially the same limited exchange. This result was correlated with the proximity of the deprotonated phosphate site to the exchangeable hydrogen atoms and the proclivity for particular nucleobase pairs to participate in nucleobase-stacking

55

Gas-Phase Hydrogen/Deuterium Exchange in Quadrupole-Ion Traps (a)

(b)

15,000

15,000

Intensity

10,000

D(8)

5000

[5´P-dAA-H]

10,000 D(2)

D(4)

5000

13C

D(7)

D(1)

13C

D(0)

0 674 675 676 677 678 679 680 681 682 683 684 685 686

0 642 643 644 645 646 647 648 649 650 651 652

Mass (m/z)

Mass (m/z)

(c)

(d)

30,000

10,000

D(6)

[5´P-dAG-H] Intensity

D(3)

D(9)

D(3)

[5´P-dGA-H]

D(2)

20,000

D(5)

10,000 D(4)

D(7) 13C

Intensity

Intensity

[5´P-dGG-H]

D(4) D(1)

5000

D(5)

D(8) D(7)

D(0)

0 658 659 660 661 662 663 664 665 666 667 668 669

13C 0 658 659 660 661 662 663 664 665 666 667 668 669

Mass (m/z)

Mass (m/z)

FIGURE 2.9 H/D-exchange mass spectra for deprotonated (a) 5′P-dGG, (b) 5′P-dAA, (c) 5′P-Dag, and (d) 5′P-dGA after 10 s exchange with D2O. Peaks are annotated as D(n) where n is the number of exchanged hydrogen atoms. Peaks labeled as 13C can be attributed solely to the isotopic contribution of the other exchanged peaks. (Reproduced from Chipuk, J.E.; Brodbelt, J.S. Int. J. Mass Spectrom, 2009 (In Press). With permission from Elsevier.)

interaction. In addition, results for the isomeric 5′-monophosphates, 5′-dAG, and 5′-dGA, were remarkably different, indicating that the H/D-exchange reaction was sequence dependent.

2.4

CONCLUSIONS

Gas-phase H/D exchange reactions offer a versatile tool for ion trap mass spectrometry. The ability of ion traps to store ions for extended periods of time and to operate at relatively high pressures makes them robust for extensive studies of H/D exchange on a variety of analytes of interest, ranging from small organic molecules to biopolymers. Elucidation of the fundamental factors that inluence gas-phase H/D exchange reactions, as well as the mechanisms, continue to be active areas of research, and it is anticipated that applications of H/D exchange will continue to expand because of the complementary information obtained compared to traditional ion-activation methods, especially when probing conformational effects in larger biopolymers.

ACKNOWLEDGMENT Funding from the Robert A. Welch Foundation (F-1155) and the National Institutes of Health (RO1 GM65956) is gratefully acknowledged.

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

REFERENCES 1. Hunt, D.F.; McEwen, C.N.; Upham, R.A. Determination of active hydrogen in organic compounds by chemical ionization mass spectrometry. Anal. Chem. 1972, 44, 1292–1294. 2. Freiser, B.S.; Woodin, R.L.; Beauchamp, J.L. Sequential deuterium exchange reactions of protonated benzenes with D2O in the gas phase by ion cyclotron resonance spectroscopy. J. Am. Chem. Soc. 1975, 97, 6893–6894. 3. Hunt, D.F.; Dethi, K. Gas-phase ion/molecule isotope-exchange reactions: methodology for counting hydrogen atoms in speciic organic structural environments by chemical ionization mass spectrometry. J. Am. Chem. Soc. 1980, 102, 6953–6963. 4. Reed, D.; Kass, S. Hydrogen-deuterium exchange at nonlabile sites: a new reaction facet with broad implications for structural and dynamic determinations. J. Am. Soc. Mass Spectrom. 2001, 12, 1163–1168. 5. Chipuk, J.E.; Brodbelt, J.S. Investigation of the gas-phase hydrogen/deuterium exchange behavior of aromatic dicarboxylic acids in a quadrupole ion trap. Int. J. Mass Spectrom. 2007, 267, 98–108. 6. Niemeyer, E.D.; Brodbelt, J.S. Isomeric differentiation of green tea catechins using gasphase hydrogen/deuterium exchange reactions. J. Am. Soc. Mass Spectrom. 2007, 18, 1749–1759. 7. DePuy, C.H. An introduction to the gas phase chemistry of anions. Int. J. Mass Spectrom. 2000, 200, 79–96. 8. Hunter, E.P.; Lias, S.G. Proton afinity data. In NIST Chemistry WebBook. NIST standard reference database No. 69; Mallard, W.G.; Linstrom, P.J., Eds.; National Institute of Standards and Technology: Gaithersburg, MD, November, 1998; http://webbook.nist.gov. 9. Ausloos, P.; Lias, S.G. Thermoneutral isotope exchange reactions of cations in the gas phase. J. Am. Chem. Soc. 1981, 103, 3641–3647. 10. Gard, E.; Grenn, M.K.; Bregar, J.; Lebrilla, C.B. Gas-phase hydrogen/deuterium exchange as a molecular probe for the interaction of methanol and protonated peptides. J. Am. Soc. Mass Spectrom. 1994, 5, 623–631. 11. Reid, G.E.; Simpson, R.J.; O’Hair, R.A.J. Probing the fragmentation reactions of protonated glycine oligomers via multistage mass spectrometry and gas-phase ion-molecule hydrogen/deuterium exchange. Int. J. Mass Spectrom. 1999, 191, 209–230. 12. Evans, S.E.; Lueck, N.; Marzluff, E.M. Gas-phase hydrogen/deuterium exchange of proteins in an ion trap mass spectrometer. Int. J. Mass Spectrom. 2003, 222, 175–187. 13. Hermann, K.; Wysocki, V.; Vorpagel, E.R. Computational investigation and hydrogen/deuterium exchange of the ixed charge derivative Tris(2,4,6-Trimethoxyphenyl) Phosphonium: implications of the aspartic acid cleavage mechanism. J. Am. Soc. Mass Spectrom. 2005, 16, 1067–1080. 14. Herrmann, K.A.; Kuppannan, K.; Wysocki, V.H. Fragmentation of doubly-protonated ion populations labeled by H/D exchange with CD3OD. Int. J. Mass Spectrom. 2006, 249–250, 93–105. 15. Zhang, J.; Brodbelt, J.S. Gas-phase hydrogen/deuterium exchange and conformations of deprotonated lavonoids and gas-phase acidities of lavonoids. J. Am. Chem. Soc. 2004, 126, 5906–5919. 16. Chipuk, J.E.; Brodbelt, J.S. Gas-phase hydrogen/deuterium exchange of 5′- and 3′-mononucleotides in a quadrupole ion trap: exploring the role of conformation and system energy. J. Am. Soc. Mass Spectrom. 2007, 18, 724–736. 17. Chipuk, J.E.; Brodbelt, J.S. Gas-phase hydrogen/deuterium exchange of dinucleotides and 5′-monophosphate dinucleotides in a quadrupole ion trap. Int. J. Mass Spectrom. 2009. In Press.

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18. Mao, D.; Ding, C.; Douglas, D.J. Hydrogen/deuterium exchange of myoglobin ions in a linear quadrupole ion trap. Rapid Commun. Mass Spectrom. 2002, 16, 1941–1945. 19. Mao, D.; Douglas, D.J. H/D exchange of gas phase bradykinin ions in a linear quadrupole ion trap. J. Am. Soc. Mass Spectrom. 2003, 14, 85–94. 20. Mao, D.; Babu, K.R.; Chen, Y-C.; Douglas, D.J. Conformations of gas-phase lysozyme ions produced from two different solution conformations. Anal. Chem. 2003, 75, 1325–1330. 21. Wright, P.J.; Zhang, J.; Douglas, D.J. Conformations of gas-phase ions of ubiquitin, cytochrome c, apomyoglobin, and β-lactoglobulin produced from two different solution conformations. J. Am. Soc. Mass Spectrom. 2008, 19, 1906–1913. 22. Chen, H.; Xu, R.; Chen, H.; Cooks, R.G.; Ouyang, Z. Ion/molecule reactions in a miniature RIT mass spectrometer. J. Mass Spectrom. 2005, 40, 1403–1411. 23. Kaltashov, I.A.; Doroshenko, V.M.; Cotter, R.J. Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer. Proteins: Struct. Funct. Genet. 1997, 28, 53–58. 24. Felix, T.; Reyzer, M.; Brodbelt, J. Hydrogen/deuterium exchange of nucleoside analogs in a quadrupole ion trap mass spectrometer. Int. J. Mass Spectrom. 1999, 191, 161–170. 25. Gronert, S. Estimation of effective ion temperatures in a quadrupole ion trap. J. Am. Soc. Mass Spectrom. 1998, 9, 845–848. 26. Wyttenbach, T.; Paizs, B.; Barran, P.; Breci, L.; Liu, D.; Suhai, S.; Wysocki, V.H.; Bowers, M.T. The effect of the initial water of hydration on the energetics, structures, and H/D exchange mechanism of a family of pentapeptides: an experimental and theoretical study. J. Am. Chem. Soc. 2003, 125, 13768–13775. 27. Dearden, D.V. KinFit: kinetics itting for coupled ordinary differential equations, version 2.0 http://chemwww.byu.edu/people/dvdearden/kinit.htm (posted April 2003). 28. Reyzer, M.L.; Brodbelt, J.S. Gas-phase H/D exchange reactions of polyamine complexes: (M + H) + , (M + alkali metal + ), and (M + 2H)2 + . J. Am. Soc. Mass Spectrom. 2000, 11, 711–721. 29. Schaaff, T.G.; Stephenson, J.L., Jr.; McLuckey, S.A. The reactivity of gaseous ions of bradykinin and its analogues with hydro- and deuteroiodic Acid. J. Am. Chem. Soc. 1999, 121, 8907–8919. 30. Schaaff, T.G.; Stephenson, J.L., Jr.; McLuckey, S.A. Gas phase H/D exchange kinetics: DI versus D2O. J. Am. Soc. Mass Spectrom. 2000, 11, 167–171.

for Multi-Stage 3 Methods Ion Processing Involving Ion/Ion Chemistry in a Quadrupole Linear Ion Trap Graeme C. McAlister and Joshua J. Coon CONTENTS 3.1 3.2

Introduction .................................................................................................... 59 Electron Transfer Dissociation (ETD) Coupled with Collision-Induced Dissociation (CID) ............................................................64 3.3 Collision-Induced Dissociation (CID) and Electron Transfer Dissociation (ETD) Coupled with Proton Transfer ........................................ 67 3.4 Ion Attachment (IA) Coupled with Collision-Induced Dissociation (CID) .......................................................................................... 70 3.5 Conclusion ...................................................................................................... 71 References ................................................................................................................ 73

3.1

INTRODUCTION

Perhaps one of the most inluential concepts in protein mass spectrometry has been the notion of enzymatic protein digestion to render a collection of peptides of suitable size for conventional tandem mass spectrometry (collision-induced dissociation, CID) [1,2]. Doubtless, this methodology has enabled signiicant progress for global protein identiication; however, many investigators now realize this approach has signiicant limitations [3]. Their conclusion is based upon the following observations. First, protein post-translational modiications (PTMs) on multi-domain proteins, and among components of protein–protein machines, work in concert; to determine their biological relevance, these patterns must be detected within the context of one another, that is to say, across the whole protein. Second, transcriptional editing processes are pervasive in higher eukaryotes and dificult to predict, even with a completely-sequenced genome. For example, three-quarters of all human proteins are expected to have at least one splice variant, that is, the gene is ‘read’ in multiple ways giving rise to multiple proteins [4–6]. Other transcription and translation events such as gene fusion, 59

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

PTR

CID nH+

ETD

Peptides and proteins

IA

FIGURE 3.1 Quadrupole ion trap mass spectrometers are capable of concatenating multiple, unique ion-processing methods during routine, robust, day-to-day operation; for example, a user can combine electron transfer dissociation (ETD), proton transfer reaction (PTR), collision-induced dissociation (CID), and ion attachment (IA) in any order in but a single scan. This ability highlights how trapped-ion instruments are capable both of mass analysis and of functioning as an ion reaction vessel.

where two separate genes are fused together to form a new unique gene, and single nucleotide polymorphisms, where two DNA molecules differ from each other by a single nucleotide, occur also. Thus, the use of short peptides as proxy markers for genes are inadequate and often misleading [7]. Today, ion trap mass spectrometers offer multiple ion-manipulation methodologies so that whole protein sequence analysis is increasingly achievable; such analysis is described as top-down proteomics [3,8–15]. With these ion-handling methods, ion trap mass spectrometers are well positioned to accelerate our ability to process effectively large species in the gas phase.* The basis of this approach is the implementation of multi-functional tools for systematic ion manipulation and processing, as depicted diagrammatically in Figure 3.1. Ion/ion chemical reactions, which include electron transfer (ET), proton transfer, and ion attachment (IA) (see below), represent one family of such tools. These technologies can be meshed with other more conventional ion trap processing methodologies such as ion isolation and CID. Together, these individual components form the foundation of, or comprise the basic toolset for, a versatile approach that promises to accelerate markedly the ield of large molecule mass spectrometry. Tandem mass spectrometry (MS/MS), in its simplest and earliest implementations on quadrupole ion traps (QITs), employed sequential (tandem-in-time MS/MS) isolation and energetic (for example, collisional) activation steps [16–22]. Modern MS/ MS experiments can involve multiple stages of mass selectivity (MSn), which employ different dissociation methods. Depending upon precursor chemistry, sometimes * See Volume 5, Chapter 4: Chemical Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural Characterization by Jennifer M. Froelich, Yali Lu, and Gavin E. Reid.

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Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry

there is no adequate activation method; in these cases, it is often advisable to combine multiple activation methods into a single concerted MSn scan type. In this chapter we discuss these concatenated MSn scan functions, focusing particularly on those scan functions that facilitate large molecule analysis. Virtually all Quadrupole ion trap (QIT)-based MS/MS experiments comprise three basic steps: (i) precursor ion isolation; (ii) precursor ion manipulation (for example, collisions, reactions such as ion/ion or ion/molecule, etc.); and (iii) product ion mass/ charge ratio analysis. The appeal of MS/MS for protein sequence analysis has come, in part, because of its ability to provide primary sequence information, and its relative ease of use when compared to alternative wet chemistry-based methods like Edman degradation [23–26]. The utility of MS/MS can often be extended by implementing multiple iterations (MSn), in which subsequent activation steps are employed to manipulate selected product ions. Due to the iterative and expandable nature of QIT-based MSn analysis, many mass-selective and/or activating steps can be strung together in a single scan; for example, one experiment utilized 11 sequential activation steps to produce an MS12 spectrum [27]. The ideal ion activation strategy would, of course, generate suficient information to identify the precursor ion in a single concerted step (that is, MS2). But, from the study of a variety of activation strategies, it has become apparent that their utility varies depending on size and charge state of the precursor ion and the presence of any labile functional groups; in some cases, one activation method is better suited for a particular subset of ions than another and, in some instances, there is no ideal activation method [28–32]. In these latter situations, coupling together multiple activation methods, to probe fully a precursor population can provide the necessary information. The scan type employed generally by ion traps relies on activation by collision, whereupon precursor ion kinetic energy is transferred to internal energy; however, as the eficacy of this process is proportional to the ratio of target (neutral) mass to projectile (ion) mass, CID is not effective for high mass ions. Gas-phase ion/ion manipulations that react cation precursors with an anionic reagent have become established in response to the development of electrospray ionization, which can produce multiply-charged ions from large polyatomic molecules, such as whole proteins [33]. The development of ion/ion chemistries from this multiple-charging capability is related directly to the fact that at least one ion/ion product must retain a charge if the product ion population is meant to be studied by mass spectrometry. The original descriptions of ion/ion chemistry focused on PTR and its application to both individual species of ions and entire ion populations [34–43]. From these original studies, the library of ion/ion reactions has expanded to include three main types: 1. Proton transfer reaction (PTR), whereby an ionic reagent either abstracts or donates a proton to a precursor [34–43]

[ M + 3H]3+ + A − → [ M + 2H]2+ + HA

(3.1);

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

2. ET, whereby an ionic reagent either abstracts or donates an electron to a precursor [44,45] [ M + 3H]3+ + A −• → [ M + 3H]2+• + A

(3.2);

3. IA, whereby the ion reagent and precursor react to form a new complex [46–49] [ M + 3H]3+ + A − → [ M + 3H + A]2+

(3.3).

Central to implementing MSn scans is the temporal lexibility allowed for by ion/ ion reactions. Following an ion/ion reaction, a completely different ion-manipulation step can be conducted nearly instantaneously, with the only required time being the few milliseconds necessary to prepare the electronics for whatever form of activation is to follow. For such experiments, ion/ion reactions offer a signiicant time advantage over ion/molecule reactions, which can require tens of seconds, or even minutes, between admission of the molecular reagent to the reaction chamber and to the completion of purging of that reagent so that the subsequent activation method may be initiated [50–60]. Technological advancement has been the primary contributor to this temporal lexibility associated with ion/ion reactions [61]. Ion/ion reactions require typically multiple ion sources for even MS2 experiments (that is, one for the cation precursor population and one for the anion reagent population), and often any additional MSn stages that require additional ion reagents may necessitate even more sources. Hence, the ability to concatenate multiple unique ion/ion activation methods during routine, robust, day-to-day operation has been made possible by the development of mass spectrometers that can accommodate simultaneously multiple ion sources. To meet these needs, researchers have developed schemes and instruments that allow ions to be injected into the QIT from multiple directions, for example, through each of an end-cap electrode and the ring electrode of a three-dimensional (3D) ion trap, in this way, two separate and distinct ion sources and ion pathways can be employed in generating and supplying the precursor ion and reagent ion populations (Figure 3.2a) [34,45,48,62–66]. Other approaches focus on adapting a single source region and ion pathway to accommodate both populations [43,67–73]. The latter scheme often requires that implementation of the sources be separated in time, that is, either the voltage applied to the sources or their placement in front of the inlet of the instrument varies during the scan cycle. In Figure 3.2b, two ESI sources are located at the MS inlet: one of the sources, a standard ESI source, is used to generate the reagent anions; the second source, a nano-spray static tip, is used to generate the precursor peptide and protein cations. The high-voltage power supplies for the two sources are triggered during the scan depending upon which ion population is needed. In a few cases, ion/ion reagents and precursors have been generated simultaneously via bipolar ionization, for example sonic spray ionization, SSI [74,75]. Ion traps themselves have been the focus of instrumentation projects. For example, linear ion traps were adapted to permit the imposition of radio-frequency (RF) voltages both

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Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry (a)

Ion trap analyzer Guard ring

Electrospray needle

Electron multiplier

Protein sample infusion

Conversion dynode Gate lens Application of high voltage DC pulser (Gate lens)

Positive ions Negative ions Needle valve

(c) A ESI + (b)

Curtain plate Orifice Skimmer

Fused silica capillary Capillary holder

Back

Center

Cl – Back lens

B Precursor ions moved to front section +

D

Triggered ± HV E Triggered ± HV

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Front lens

C Nano-ESI tip

ASGDI sample containment vessel

F

0V –10 V +5 V

Anion injection

+

+

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–

–

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+ –

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+

–

0V

FIGURE 3.2 Multiple methods are used to generate and to inject reagent ions for conducting ion/ion reactions in a quadrupole ion trap. (a) Schematic of a quadrupole ion trap, which was adapted to permit the injection of reagent ions through the ring electrode (Reproduced from Stephenson, J.L.; McLuckey, S.A. Int. J. Mass Spectrom. Ion Processes. 1997, 162, 89–106. With permission from Elsevier.) (b) Method of injection of cations and reagent anions via dual atmospheric pressure ion emitters; this approach requires very little, if any, modiication of the ion trap system (Reproduced from Xia, Y.; McLuckey, S.A. J. Am. Soc. Mass Spectrom. 2008, 19, 173–189. With permission from Elsevier.) (c) A schematic diagram illustrating the implementation of ion/ion reactions on a quadrupole linear ion trap system. Note reagent anions are generated on the right side of the diagram via a CI source. This quadrupole linear ion trap system has been adapted such that either it can mix selectively ions of opposite polarity or it can segregate ions of opposite polarity via a DC offset and charge-sign independent trapping. (Reproduced from Syka, J.E.P., Coon, J.J.; Schroeder, M.J.; Shabanowitz, J.; Hunt, D.F., Proc. Natl. Acad. Sci. 2004, 101, 9528–9533. With permission from the National Academy of Sciences.)

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

radially and axially thereby permitting charge-sign independent trapping, which in turn allows the device to function as an ion/ion reaction vessel* (Figure 3.2c) [45]. In this chapter, we present details of how the compilation of ion-manipulation methodologies in QIT mass spectrometers can eliminate many of the restrictions that limit currently large molecule mass spectrometry. Speciically, we highlight three MSn interrogation schemes that show genuine promise to enable whole protein analysis: (i) ETD coupled with CID; (ii) CID and ETD coupled with Proton Transfer; and (iii) IA coupled with CID.

3.2

ELECTRON TRANSFER DISSOCIATION (ETD) COUPLED WITH COLLISION-INDUCED DISSOCIATION (CID)

Electron-based dissociation methods (ETD and its forerunner electron capture dissociation, ECD) are highly eficient at generating c- and z• -type fragment ions from peptide and whole protein precursor ions via a process that is essentially independent of peptide length, amino acid composition, and post-translation modiication (PTM) state [28,45,76–81]. Precursor charge state and m/z-value are important factors, however, that can affect the dissociation eficiency of ETD methods [82–85]. The most widely-held explanation for why lowly-charged, high-m/z ions do not fragment eficiently by ETD is that there is a charge-to-residue threshold to eficient ETD, that is, a minimum charge density is necessary in order to achieve direct dissociation [85,86]. If a particular precursor ion falls below this threshold then, following cleavage of the backbone bond, the probability that the two product ions remain non-covalently bound increases. To obviate this problem more energy can be added to the post-reaction product, for example for ECD, analyte ions can be activated by infrared photons prior to ECD, a process that has been termed activated ion ECD (AI-ECD) [10,14,87–90]. The additional energy absorbed disrupts preferentially the non-covalent bonds and increases fragmentation eficiency. The QIT-based ion/ion analog of AI-ECD was described irst by Swaney et al. [32]. There we described in detail how gentle CID of the non-dissociative ET products (ETnoD) could increase signiicantly the yield of c- and z• -type product ions; this fragmentation process is referred to as EtcaD in Ref. [32]. Optimization of the energy was essential for the preferential breakage of the non-covalent bonds because, if too much energy was imparted, then b- and y-type product ions were formed. Over a large data set, that is, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a complex mixture, we showed how data-dependent activation of the ETnoD products by these optimal CID parameters increased the overall number of c- and z• -type ions produced, thereby ETD eficiency was increased as well (Figure 3.3). Since this original description of the ETD/CID MS/MS scan type (ETcaD), it has been implemented on commercial instruments, applied toward biologically-relevant samples, and expanded upon [28,85,91,92]. For example, Han et al. [92] coupled ETD with beam-type CID on their QTRAP hybrid triple quadrupole/linear ion trap instrument (Figure 3.4). An advantage of both this implementation, and beam-type CID in general, is that the spectra do not suffer from the low mass cut-off that is * See Volume 5, Chapter 1: Ion/Ion Reactions in Electrodynamic Ion Traps by Jian Liu and Scott A. McLuckey.

ETD, 532 m/z

300

400

z+3

z+12

1000

1000

1200

1400

z+13

(M+2H)++

400

200

400

Intensity: 1.0×105 z3

z4 600

z5 c+ 5 z6

500

z+5 700

800 m/z

z7

z9

400

600

800

1000 m/z

×4

1200

(Precursor)

1400

1600

1800

c20

z+20

2000

×8

200

z4 400

Intensity: 1.0×104

ET-CID,966 m/z

600

z5

z6

Y6

800

z7 z8

1000 m/z

z9

1200

1400

(Precursor) z ×4 z 12 11 z 13 z14 z13

(M+2H)++

1600

z16

1800

c19 2000

(M+2H)++ z+ c+20 20

1400

z+13

(M+2H)++

1000

(M+2H)++

FIGURE 3.3 Comparison of ETD (a–c) and ETcaD (d–f) product ion mass spectra (single scan) for three tryptic peptides. Each product ion mass spectrum was acquired during a data-dependent analysis of a complex tryptic peptide mixture derived from Arabidopsis. (Reproduced from Swaney D.L.; McAlister, G.C.; Wirtala, M.; Schwartz, J.C.; Syka, J.E.P.; Coon, J.J., Anal. Chem. 2007, 79, 477–485. With permission from the American Chemical Society.)

200

Intensity: 1.0×104

ETD, 966 m/z

(M+2H)++

1200

z+12 c12

c13

z8 z+ 8 900

z+11

z+7

c11

800

z+10

z+6

1000

(Precursor) ×2 z8

(M+2H)++

600 m/z

z4 ×1.5

(Precursor)

(M+2H)++

VG P P PA P S GG L P GT DN SD QA R

800 m/z

11

ET-CID,532 m/z

300

z3

I G S E I S S LT L E E A R

(e)

200

Intensity: 1.0×105 z2

VV D IV D TF R

ET-CID,532 m/z

VG P P PA P S GG L P GT DN SD QA R

600

z

+

900

(Precursor) ×3

z+10

800

z+8

×8.5

700

z+7

(M+2H)++

5

6

(M+2H)++

600 m/z

z

+

z

+

×5

(d)

(f )

400

500

z+4

(Precursor)

(M+2H)++

(M+2H)++

(c)

200

Intensity: 1.0×105

I G S E I S S LT L E E A R

ETD, 753 m/z

(b)

200

Intensity: 1.0×105

VV D IV D TF R

(a)

Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry 65

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

Nanospray emitter Q0

IQ2 ~ IQ3 ~ Q2 Q1 Q3

Pulsed + HV Pulsed Reagent vapor – HV APCI emitter

FIGURE 3.4 Schematic diagram of the QTRAP hybrid triple quadrupole/linear ion trap instrument that has been modiied to conduct ion/ion reactions in Q2. (Reproduced from Han, H.L.; Xia, Y.; McLuckey, S.A., Rapid Commun. Mass Spectrom. 2007, 21, 1567–1573. With permission from John Wiley & Sons, Inc.)

characteristic of ion trap CID. Also, multiple ETnoD product ions can be activated simultaneously instead of sequentially, as would be required with ion trap CID. An analog to coupling ETD with CID, which uses CID as the primary dissociation mechanism, is the addition of a PTR activation step prior to CID. Like ETD, CID activation is also charge dependent. But unlike ETD, where fragmentation eficiency increases with increasing charge density, CID fragmentation eficiency tends to be higher for lowly-charged precursor ions [93–95]. Unfortunately, during typical proteomics experiments, ionization mechanisms determine almost exclusively precursor charge state, and larger precursors tend to ionize through the acquisition of more charges. To some degree, these trends obstruct the interrogation of all large precursor ions via CID. The inclusion of a PTR activation step prior to CID can help mitigate this problem. As noted earlier, PTR involves anionic reagents that abstract protons from cationic precursors [34–43]. In the context of this experiment, a PTR step helps by stripping away charges from a precursor, that is too highly-charged to be dissociated effectively via CID, and places it in a charge state where it is more amenable to fragmentation [96]. An interesting variant of this experiment involves performing ion parking, pioneered by McLuckey et al. [96], during the PTR ion/ion reaction. Ion parking is the quenching of an ion/ion reaction at a speciic product ion mixture instead of letting those products react further with additional reagent ions. Ion parking can take two forms: (i) separation of the reagents and products in space via segregation of the ion clouds; and (ii) excitation of the product ions via the application of a supplemental waveform, which prevents the oppositely-charged ions from forming an orbiting complex, a prerequisite to an ion/ion reaction [13,97–102]. In either case, the inclusion of a parking step allows all of the precursor ions to be converted eficiently from one or multiple charge states to product ions of a lower-charge state. As noted earlier, the ideal mass spectrometer-based activation method would require only a single step. However, as we have learned more about ion activation methods, both their abilities and their limitations, we have arrived at the conclusion that no single ion activation method is best suited for interrogating every possible precursor ion. Hence, the experiments discussed here highlight the utility of mixing multiple activation methods in a single scan as a viable alternative; when CID is combined with ETD, ETD-like dissociation is observed wherein rich product ion mass spectra permit identiication of precursor ions that have low charge states and m/z-values.

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3.3

67

COLLISION-INDUCED DISSOCIATION (CID) AND ELECTRON TRANSFER DISSOCIATION (ETD) COUPLED WITH PROTON TRANSFER (PTR)

By analogy with the successful coupling of ETD with CID, wherein the second activation step augmented the irst activation step by converting irst generation product ions into new species that are more informative of sequence, CID, and ETD have been coupled to PTR [36,77]. QITs are capable of high-sensitivity analysis and fast duty cycles (spectra s−1) at unit mass-resolving power. This limited resolution generally means that only singly and doubly-charged ions can be identiied. Operation of QITs at resolutions suficient to determine fragment ion charges greater than two is possible, but generally is considered impractical as the scan rates and sensitivity are substantially reduced [103]. Principally as a result of this limitation, MS/MS analysis on QITs has been restricted to lowly-charged precursor ions (for example, in bottom-up proteomics), as these populations tend to produce lowly-charged product ions that are ideal for QIT analysis. Proton transfer (PTR), one of the ion/ion reactions detailed above, has been developed primarily as a means of simplifying mixtures of highly-charged ions [34–43]. Higher-charged precursor ions will react faster than lower-charge precursor ions as reaction rates for an ion/ion reactions increase by the square of the charge state of the participating ions [35,104,105]. Therefore, PTR activation of a mixture of highlycharged ions will result in the ion population being concentrated into lower-charge states. The utility then of PTR, as it applies to interrogation of larger highly-charged precursor peptides and proteins, is that following the initial activation and dissociation (MS2) of the precursor cation, the product population can be simpliied and concentrated en masse into lower-charge state ions by a subsequent PTR activation step. Initially, this multi-stage activation scheme employed CID as the prime activation step (MS2) [36]. These experiments, pioneered by Stephenson and McLuckey, were carried out on a Finnigan 3D QIT, which was modiied to allow for injection of PTR reagent anions, which were generated via glow discharge of the sampled headspace of perluoro-1,3-dimethyl-cyclohexane (PDCH), through an additional hole in the ring electrode. Cation precursors, generated via electrospray, were injected through a hole in one of the end-cap electrodes. Highlighted in Figure 3.5 is the interrogation of melittin via CID/PTR. The mass spectrum in Figure 3.5a is the post-CID MS/MS spectrum of the [M + 4H]4 + melittin ion, and Figure 3.5b displays the same ion population after being subjected to a PTR reaction for 100 ms. Following PTR activation, the b- and y-type ions are concentrated in their lower-charge states. In this example, the coupling of CID with PTR allowed for the successful interrogation of a higher-charged precursor species by removing charge from the multiply-protonated CID product ions. ET dissociation is coupled easily also with a subsequent PTR step. Even though dissociation via ETD is inherently destructive to precursor charges, in that typically one charge is neutralized for every backbone bond that is broken, in general, this procedure does not lead inevitably to a reduction of the aggregate charge of the ion population to the point that would permit isotopic resolution for identiication. For highly-charged precursor ions, multiple ion/ion reactions are required in order to achieve the desired level of charge reduction; however, multiple ETD reactions will result in multiple breakages of the peptide backbone bonds which, in turn, produce

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a)

Intensity (arbitrary units)

12000

2+ y13

8000

[M+4H–2NH3]4+ [M+4H]4+ 3+ y13

4000

3+ y24

2+; b+ y21 13 3+ y17

b5+

+

b12

0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z (b) G I G A V L K V L T T G L P A L I S W I K R K R Q Q–NH2

Intensity (arbitrary units)

1600

+ y13

1200 2+ y13

800

400

b+8 b+7

+ y14

+ b10

b+9

+

+ b + b12 13 y11 + + y10 y12 b+ 11

[M+H–NH3]+ + y16

+ y15

[M+H–2NH3]+ + y19 + y17 y+ y+ + 21 + 24 + y18 y20 y22

0 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z

FIGURE 3.5 (a) Pre-ion/ion reaction (PTR) MS/MS spectrum of the [M + 4H]4 + parent ion of melittin. (b) Post-ion/ion reaction (PTR) MS3 spectrum of the [M + 4H]4 + parent ion of melittin. (Reproduced from Stephenson, J.L.; McLuckey, S.A. Anal. Chem. 1998, 70, 3533–3544. With permission from the American Chemical Society.)

internal fragments that will convolute substantially the product ion mass spectrum. Hence, two types of ion/ion reactions are required; one to break the backbone bonds and one to strip away the excess charges. The irst reported use of the ETD/PTR scan type involved reactions between cation precursors with radical luoranthene anions (ETD), followed by reactions of cationic ETD product ions with benzoic acid anions (PTR) (Figure 3.6) [77]. These

69

Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry (a) 15 ms ETD

Int: 1.2 × 104

15 ms ETD/50 ms PTR

Int: 9.0 × 103

15 ms ETD/100 ms PTR

Int: 4.6 × 103

(b)

(c)

(d) 15 ms ETD/150 ms PTR c2 c3 c4 c7 c5 z8 z3 z4 z5 z6 z7 c6 400

600

800

z17++ c17++ c8

Int: 3.4 × 103

c8

c17/z17 c10 z13 c15/z15 z15 c9 c c z9 z10 z11c11z12 12 c13 z1414 c16

1000

1200 m/z

1400

1600

1800

2000

(e) M Q I F V KT LT G K T I TL E V E S S D T ID N V K S K IQ D K E G I P P D Q Q R L I F A G KQ L E D G R T L S D Y N I Q K E S T LH L V L R L R G G

FIGURE 3.6 Product ion mass spectrum of ubiquitin generated by sequential ion/ion reactions. (a) Whole protein dissociation (ubiquitin + 13, having 13 residues, that is, 13 amino-acids, m/z 659) after reacting for 15 ms with the radical anion of luoranthene. Note production of several hundred highly-charged unresolved c- and z-type product ions. (b–d), The subsequent reaction of these product ions with even-electron anions of benzoic acid for 50 (b), 100 (c), and 150 (d) ms. Note the gradual degradation of multiply-charged products, leaving predominately doubly and singly-charged fragments after 150 ms. (e) The resulting sequence coverage considering only singly-charged product ions. Each mass spectrum is the average of 50 spectra (30-s acquisition), and the relative ion abundance is indicated on the ordinate. (Reproduced from Coon, J.J.; Ueberheibe, B.; Syka, J.E.P.; Dryhurst, D.D.; Ausio, J.; Shabanowitz, J.; Hunt, D.F., Proc. Natl. Acad. Sci. 2005, 102, 9463–9468.)

experiments were conducted on a modiied Finnigan LTQ mass spectrometer. The instrument was adapted to accommodate a chemical ionization (CI) source on the far side of the linear ion trap relative to the standard atmospheric pressure source, and the electronics supplying the linear ion trap were modiied to allow for the

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

superimposition of an RF voltage on the end lenses, which allows for charge-sign independent trapping. Both luoranthene (for ETD) and benzoic acid (for PTR) were volatilized and ionized in the added CI source (that is, one source was used to generate both reagent anions); depending upon which ion/ion activation method was being employed, the competing ion/ion reagent was removed by the application of a selective waveform. In Figure 3.6, four mass spectra are presented, each one utilizing the same ETD reaction parameters but for progressively longer PTR times (0–100 ms, in Figure 3.6A through D, respectively). As both sets of authors noted in these papers, by deconvoluting the charge states of the product ions using an additional PTR step, an experimenter can extend signiicantly the size range of precursor ions that are compatible with QIT instruments [36,77]. The propensity of researchers to perform bottom-up proteomics experiments involving smaller precursor peptides is not the result of a lack of interest in interrogating longer precursor peptides and whole proteins. Since the advent of these scan types, which involve ion dissociation followed by PTR, they have been employed in studies to investigate biological problems such as histone PTM state and to identify intact proteins [106,107]. Identiication of PTM motifs and the importance of alternative splicing on biological systems require the analysis of at least large peptides and, preferably, intact proteins.

3.4

ION ATTACHMENT (IA) COUPLED WITH COLLISION-INDUCED DISSOCIATION (CID)

In the two previously-described experiments, the second activation step augmented the irst activation step by converting the irst generation product ions into new species that were more informative of sequence; for example, PTR was coupled to CID to convert the CID product ions, which were produced from the interrogation of high-charge state precursors, into forms that were better suited for QIT analysis. But for both the MS2 and MS3 scan types, the dominant fragmentation pathways were the same, that is, the inclusion of PTR did not result in any additional backbone bonds being broken. Coupling an IA reaction to CID however, is done for tangential reasons, that is, IA is included before the CID step to bias the CID process toward forming different product ions [49]. The goal is still to produce sequence-informative ions that would not have been possible using only an MS2 scan type but, in this case, the production of sequenceinformative ions is achieved by biasing the CID process away from generating one set of product ions and toward the generation of a completely different set. To date there has been very little published about IA reactions. A few metal-based anions, phosphorus hexaluoride, and I¯ have all been reported to form complexes with peptide cations [37,108,109]. Yet upon CID, these complexes tend to separate easily, which implies that they are simply coming together to form a long-lived intermediate of the PTR pathway. Glish and Payne reported the irst IA work detailing how activation with FeCO −2 resulted in peptide fragment ions that contained the anion reagent, which implies that the anion forms bond with the peptide that are stronger than the peptide’s own intramolecular backbone bonds [48]. Also, Gunawardena et al. showed that a reaction between AuCl −2 and peptide cations resulted in selective cleavage of disulide bonds [46].

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Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry

O H3N+

N H

+

O

3.

O

R

HN

O O –

Re N O

NH O N –2 O + Re O N OH

H

N

ÖH O

OH OH

OH

+

N O

O

H

O O

OH

5. O

R+

+

R +

O NH + 3

Re– N

+

NH3

N

N

H2O O

O –2 Re OH O

4. O

+

N H

R+

O

O

H N

H3N+

OH N H

O

R+ NH3

O

H N

2.

O ÖH

Ö

O – Re O

1.

O

O

+

O

H2O O

OH

FIGURE 3.7 Proposed mechanism for the attachment of rhenate to a doubly-protonated peptide. (Reproduced from McAlister, G.C.; Kiessel, S.E.B.; Coon, J.J., Int. J. Mass Spectrom. 2008, 276, 149–152. With permission from Elsevier).

Recently, we demonstrated that reactions between rhenate Re O3− and multiplycharged peptide cations resulted in a new compound that, upon CID, retained the metal oxide (Figure 3.7) [49]. These experiments were conducted on a modiied Finnigan LTQ mass spectrometer, which was adapted to accommodate a CI source. The Re O3− was generated initially by leaking atmospheric air into the CI source region, and, later, by leaking in high-purity 18O2. In both cases, molecular oxygen reacted with the rhenium ilament of the CI source to generate the anion reagent. Of particular interest to us, the presence of rhenate alters the preferred CID fragmentation pathways (Figure 3.8). Upon CID, the singly-charged synthetic peptide RAAAKAAAK produces the b8+  ion; however, following the addition of a Re O3− ion to the doubly-charged species via IA, CID produces fragment ions derived from four different backbone bond cleavages. One exciting possible application of this MS3 scan type, which involves an IA reaction followed by CID, is site-speciic gas phase disassembly of peptide and protein cations. In this application, anion reagents would bind to the peptide or protein with a high degree of site or motif speciicity. Then during the following dissociation step, the bound anion reagent would direct backbone cleavage. In this manner, a large protein could be broken apart systematically and interrogated in much smaller chunks, which are more readily sequenced by the instrument. The anions presented in this section, which are capable of forming covalently-bound complexes with cation precursors, represent a solid irst step; however, they are not at the level of functionality described above.

3.5

CONCLUSION

The perfect ion activation technique would produce a sequence-informative spectrum in a single concerted step (that is, MS2) regardless of peptide length, amino acid

72

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a) [M+H]+

b8+H2O [M+H]+ (Precursor)

R A A A KA A A K

b8

[M+H–H2O]+

(b) [M+2H+ReO3–3H2O]+

[M+2H+ReO3–H2O]+

RAAAKAAAK a4+ReO

b4+ReO a8+ReO a7+ReO b +ReO b8+ReO 7

a3+ReO 300

400

500

600

700 m/z

800

900

1000

FIGURE 3.8 Collision-induced dissociation (CID) of the singly-protonated peptide RAAAKAAAK (a) and the same peptide following attachment of the rhenate anion (b). Note the preferred dissociation pathways change. (Reproduced from McAlister, G.C.; Kiessel, S.E.B.; Coon, J.J., Int. J. Mass Spectrom. 2008, 276, 149–152. With permission from Elsevier.)

composition, or presence of PTMs. Unfortunately, there are niches of peptide and protein precursors, delineated by size, charge, amino acid composition, and PTM state, for which no ideal activation method exists. As a means of accommodating this diverse set of precursors, selected activation methods can be brought together in multiple-stage activation schemes (MSn) that are tailored suficiently to the precursor ion chemistry to allow for characterization of the sequence. In this vein, ion/ion reactions allow for a variety of ion manipulations that are otherwise not possible, are coupled easily with CID, and with each other in discrete reaction sequences. The three scan types described here are by no means a complete listing of all the possible MSn scan types which contain ion/ion reactions. For example, McLuckey and coworkers have published extensively on charge inversion reactions and their utility in MSn scans as a means of dissociating precursor polarity from the ionization process; for example, precursors can be ionized in negative mode but activated in a positive charge state following charge inversion [46,73,110–112]. There have been studies that have examined the affect of CID on speciic ETD fragment ions [113]. For every ion/ ion reaction presented here there are numerous different reagent analogs, each possessing a unique set of drawbacks and beneits, which can and in many cases have been used. As trapped-ion mass spectrometers capable of ion/ion reactions have become more prevalent and accessible to the scientiic community, the body of knowledge covering ion/ion chemistries has grown accordingly. This rapid increase in knowledge has been transformative in the sense that these instruments have undergone a transition from simple mass spectrometers into gas-phase chemical reactors of extraordinary

Methods for Multi-Stage Ion Processing Involving Ion/Ion Chemistry

73

performance. For example, such gas-phase ion-trapping devices have evolved to the point where multi-stage reactions can be performed in milliseconds, reaction parameters such as time and reagent population size can be manipulated easily, and single product ion detection has been realized. In touching lightly on these attributes of gas-phase ion-trapping devices, this chapter has described merely the irst stages of this exciting transition of enormous potential.

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15. Forbes, A.J.; Patrie, S.M.; Taylor, G.K.; Kim, Y.B.; Jiang, L.H.; Kelleher, N.L. Targeted analysis and discovery of posttranslational modiications in proteins from methanogenic archaea by top-down MS. Proc. Natl. Acad. Sci. 2004, 101, 2678–2683. 16. Brodbelt, J.S.; Kenttamaa, H.I.; Cooks, R.G. Energy-resolved collisional activation of dimethyl phosphonate and dimethyl phosphite ions in a quadrupole ion trap and a triple quadrupole mass-spectrometer. Org. Mass Spectrom. 1988, 23, 6–9. 17. Louris, J.N.; Brodbelt, J.S.; Cooks, R.G. Photodissociation in a quadrupole ion trap mass-spectrometer using a iber optic interface. Int. J. Mass Spectrom. Ion Processes 1987, 75, 345–352. 18. Louris, J.N.; Cooks, R.G.; Syka, J.E.P.; Kelley, P.E.; Stafford, G.C.; Todd, J.F.J. Instrumentation, applications, and energy deposition in quadrupole ion-trap tandem mass spectrometry. Anal. Chem. 1987, 59, 1677–1685. 19. McLuckey, S.A.; Glish, G.L.; Kelley, P.E. Collision-activated dissociation of negative-ions in an ion trap mass-spectrometer. Anal. Chem. 1987, 59, 1670–1674. 20. Stafford, G.C.; Kelley, P.E.; Syka, J.E.P.; Reynolds, W.E.; Todd, J.F.J. Recent improvements in and analytical applications of advanced ion trap technology. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85–98. 21. Strife, R.J.; Kelley, P.E.; Weber-Grabau, M. Tandem mass spectrometry of prostaglandins: A comparison of an ion trap and a reversed geometry sector instrument. Rapid Commun. Mass Spectrom. 1988, 2, 105–109. 22. Syka, J.E.P.; Louris, J.N.; Kelley, P.E.; Stafford, G.C.; Reynolds, W.E. Method of operating ion trap detector in MS/MS mode. U.S. Patent 1988, 4,736,101. 23. Sadygov, R.G.; Cociorva, D.; Yates, J.R. Large-scale database searching using tandem mass spectra: Looking up the answer in the back of the book. Nature Methods 2004, 1, 195–202. 24. LeDuc, R.D.; Taylor, G.K.; Kim, Y.B.; Januszyk, T.E.; Bynum, L.H.; Sola, J.V.; Garavelli, J.S.; Kelleher, N.L. ProSight PTM: An integrated environment for protein identiication and characterization by top-down mass spectrometry. Nucleic Acids Research 2004, 32, W340–W345. 25. Perkins, D.N.; Pappin, D.J.C.; Creasy, D.M.; Cottrell, J.S. Probability-based protein identiication by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–3567. 26. Eng, J.K.; McCormack, A.L.; Yates, J.R. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994, 5, 976–989. 27. Louris, J.N.; Brodbeltlustig, J.S.; Cooks, R.G.; Glish, G.L.; Vanberkel, G.J.; McLuckey, S.A. Ion isolation and sequential stages of mass-spectrometry in a quadrupole ion trap mass-spectrometer. Int. J. Mass Spectrom. Ion Processes 1990, 96, 117–137. 28. Molina, H.; Matthiesen, R.; Kandasamy, K.; Pandey, A. Comprehensive comparison of collision induced dissociation and electron transfer dissociation. Anal. Chem. 2008, 80, 4825–4835. 29. Zubarev, R.A.; Zubarev, A.R.; Savitski, M.M. Electron capture/transfer versus collisionally activated/induced dissociations: Solo or duet? J. Am. Soc. Mass Spectrom. 2008, 19, 753–761. 30. Good, D.M.; Wirtala, M.; McAlister, G.C.; Coon, J.J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 2007, 6, 1942–1951. 31. Swaney, D.L.; McAlister, G.C.; Coon, J.J. Decision tree-driven tandem mass spectrometry for shotgun proteomics. Nature Methods 2008, 5, 959–964. 32. Swaney, D.L.; McAlister, G.C.; Wirtala, M.; Schwartz, J.C.; Syka, J.E.P.; Coon, J.J. Supplemental activation method for high-eficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 2007, 79, 477–485.

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53. Custer, T.G.; Kato, S.; Bierbaum, V.M.; Howard, C.J.; Morrison, G.C. Gas-phase kinetics and mechanism of the reactions of protonated hydrazine with carbonyl compounds. Gas-phase hydrazone formation: Kinetics and mechanism. J. Am. Chem. Soc. 2004, 126, 2744–2754. 54. Gronert, S.; Huang, R.; Li, K.H. Gas phase derivatization in peptide analysis I: The utility of trimethyl borate in identifying phosphorylation sites. Int. J. Mass Spectrom. 2004, 231, 179–187. 55. Gao, H.; Petzold, C.J.; Leavell, M.D.; Leary, J.A. Investigation of ion/molecule reactions as a quantiication method for phosphorylated positional isomers: An FT-ICR approach. J. Am. Soc. Mass Spectrom. 2003, 14, 916–924. 56. Milman, B.L. Cluster ions of diquat and paraquat in electrospray ionization mass spectra and their collision-induced dissociation spectra. Rapid Commun. Mass Spectrom. 2003, 17, 1344–1349. 57. Leavell, M.D.; Leary, J.A. Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS. J. Am. Soc. Mass Spectrom. 2003, 14, 323–331. 58. Gronert, S.; O’Hair, R.A.J. Gas phase reactions of trimethyl borate with phosphates and their non-covalent complexes. J. Am. Soc. Mass Spectrom. 2002, 13, 1088–1098. 59. Leavell, M.D.; Kruppa, G.H.; Leary, J.A. Analysis of phosphate position in hexose monosaccharides using ion-molecule reactions and SORI-CID on an FT-ICR mass spectrometer. Anal. Chem. 2002, 74, 2608–2611. 60. Green, M.K.; Lebrilla, C.B. Ion-molecule reactions as probes of gas-phase structures of peptides and proteins. Mass Spectrom. Rev. 1997, 16, 53–71. 61. Xia, Y.; McLuckey, S.A. Evolution of instrumentation for the study of gas-phase ion/ion chemistry via mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19, 173–189. 62. Hogan, J.M.; Pitteri, S.J.; Chrisman, P.A.; McLuckey, S.A. Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation. J. Proteome Res. 2005, 4, 628–632. 63. Herron, W.J.; Goeringer, D.E.; McLuckey, S.A. Product ion charge state determination via ion/ion proton transfer reactions. Anal. Chem. 1996, 68, 257–262. 64. Herron, W.J.; Goeringer, D.E.; McLuckey, S.A. Gas-phase electron-transfer reactions from multiply-charged anions to rare-gas cations. J. Am. Chem. Soc. 1995, 117, 11555–11562. 65. Herron, W.J.; Goeringer, D.E.; McLuckey, S.A. Ion-ion reactions in the gas-phase-protontransfer reactions of protonated pyridine with multiply-charged oligonucleotide anions. J. Am. Soc. Mass Spectrom. 1995, 6, 529–532. 66. Berberich, D.W.; Yost, R.A. Negative chemical-ionization in quadrupole ion-trap massspectrometry-effects of applied voltages and reaction-times. J. Am. Soc. Mass Spectrom. 1994, 5, 757–764. 67. McAlister, G.C.; Phanstiel, D.; Good, D.M.; Berggren, W.T.; Coon, J.J. Implementation of electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer. Anal. Chem. 2007, 79, 3525–3534. 68. Williams, D.K.; McAlister, G.C.; Good, D.M.; Coon, J.J.; Muddiman, D.C. Dual electrospray ion source for electron-transfer dissociation on a hybrid linear ion traporbitrap mass spectrometer. Anal. Chem. 2007, 79, 7916–7919. 69. Gunawardena, H.P.; McLuckey, S.A. Synthesis of multi-unit protein hetero-complexes in the gas phase via ion-ion chemistry. J. Mass Spectrom. 2004, 39, 630–638. 70. Wells, J.M.; Chrisman, P.A.; McLuckey, S.A. Formation and characterization of proteinprotein complexes in vacuo. J. Am. Chem. Soc. 2003, 125, 7238–7249. 71. Badman, E.R.; Chrisman, P.A.; McLuckey, S.A. A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions. Anal. Chem. 2002, 74, 6237–6243.

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72. Wells, J.M.; Chrisman, P.A.; McLuckey, S.A. “Dueling” ESI: Instrumentation to study ion/ion reactions of electrospray-generated cations and anions. J. Am. Soc. Mass Spectrom. 2002, 13, 614–622. 73. Xia, Y.; Liang, X.R.; McLuckey, S.A. Pulsed dual electrospray ionization for ion/ion reactions. J. Am. Soc. Mass Spectrom. 2005, 16, 1750–1756. 74. Xia, Y.; Liang, X.R.; McLuckey, S.A. Sonic spray as a dual polarity ion source for ion/ ion reactions. Anal. Chem. 2005, 77, 3683–3689. 75. Takats, Z.; Nanita, S.C.; Cooks, R.G.; Schlosser, G.; Vekey, K. Amino acid clusters formed by sonic spray ionization. Anal. Chem. 2003, 75, 1514–1523. 76. Coon, J.J.; Syka, J.E.P.; Shabanowitz, J.; Hunt, D.F. Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques 2005, 38, 519–523. 77. Coon, J.J.; Ueberheide, B.; Syka, J.E.P.; Dryhurst, D.D.; Ausio, J.; Shabanowitz, J.; Hunt, D.F. Protein identiication using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. 2005, 102, 9463–9468. 78. Hauser, N.J.; Han, H.L.; McLuckey, S.A.; Basile, F. Electron transfer dissociation of peptides generated by microwave D-cleavage digestion of proteins. J. Proteome Res. 2008, 7, 1867–1872. 79. Gunawardena, H.P.; Gorenstein, L.; Erickson, D.E.; Xia, Y.; McLuckey, S.A. Electron transfer dissociation of multiply protonated and ixed charge disulide linked polypeptides. Int. J. Mass Spectrom. 2007, 265, 130–138. 80. Liang, X.R.; Hager, J.W.; McLuckey, S.A. Transmission mode ion/ion electron-transfer dissociation in a linear ion trap. Anal. Chem. 2007, 79, 3363–3370. 81. Huang, T.Y.; Emory, J.F.; O’Hair, R.A.J.; McLuckey, S.A. Electron-transfer reagent anion formation via electrospray ionization and collision-induced dissociation. Anal. Chem. 2006, 78, 7387–7391. 82. Iavarone, A.T.; Paech, K.; Williams, E.R. Effects of charge state and cationizing agent on the electron capture dissociation of a peptide. Anal. Chem. 2004, 76, 2231–2238. 83. Pitteri, S.J.; Chrisman, P.A.; McLuckey, S.A. Electron-transfer ion/ion reactions of doubly protonated peptides: Effect of elevated bath gas temperature. Anal. Chem. 2005, 77, 5662–5669. 84. Zubarev, R.A.; Horn, D.M.; Fridriksson, E.K.; Kelleher, N.L.; Kruger, N.A.; Lewis, M.A.; Carpenter, B.K.; McLafferty, F.W. Electron capture dissociation for structural characterization of multiply charged protein cations. Anal. Chem. 2000, 72, 563–573. 85. Good, D.M.; Wirtala, M.; McAlister, G.C.; Coon, J.J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 2007, 6, 1942–1951. 86. Olsen, J.V.; Haselmann, K.F.; Nielsen, M.L.; Budnik, B.A.; Nielsen, P.E.; Zubarev, R.A. Comparison of electron capture dissociation and collisionally activated dissociation of polycations of peptide nucleic acids. Rapid Commun. Mass Spectrom. 2001, 15, 969–974. 87. Hakansson, K.; Chalmers, M.J.; Quinn, J.P.; McFarland, M.A.; Hendrickson, C.L.; Marshall, A.G. Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer. Anal. Chem. 2003, 75, 3256–3262. 88. Sze, S.K.; Ge, Y.; Oh, H.B.; McLafferty, F.W. Plasma electron capture characterization of large dissociation for the proteins by top down mass spectrometry. Anal. Chem. 2003, 75, 1599–1603. 89. Shi, S.D.H.; Hemling, M.E.; Carr, S.A.; Horn, D.M.; Lindh, I.; McLafferty, F.W. Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry. Anal. Chem. 2001, 73, 19–22. 90. Horn, D.M.; Ge, Y.; McLafferty, F.W. Activated ion electron capture dissociation for mass spectral sequencing of larger (42 kDa) proteins. Anal. Chem. 2000, 72, 4778–4784.

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91. Xia, Y.; Han, H.; McLuckey, S.A. Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions. Anal. Chem. 2008, 80, 1111–1117. 92. Han, H.L.; Xia, Y.; McLuckey, S.A. Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer. Rapid Commun. Mass Spectrom. 2007, 21, 1567–1573. 93. Watson, D.J.; McLuckey, S.A. Charge state dependent ion trap collision-induced dissociation of reduced bovine and porcine trypsin cations. Int. J. Mass Spectrom. 2006, 255, 53–64. 94. Pitteri, S.J.; Chrisman, P.A.; Badman, E.R.; McLuckey, S.A. Charge-state dependent dissociation of a trypsin/inhibitor complex via ion trap collisional activation. Int. J. Mass Spectrom. 2006, 253, 147–155. 95. Wells, J.M.; McLuckey, S.A. Collision-induced dissociation (CID) of peptides and proteins. Biol. Mass Spectrom. 2005, 402, 148–185. 96. Liu, J.; Chrisman, P.A.; Erickson, D.E.; McLuckey, S.A. Relative information content and top-down proteomics by mass spectrometry: Utility of ion/ion proton-transfer reactions in electrospray-based approaches. Anal. Chem. 2007, 79, 1073–1081. 97. Chrisman, P.A.; Pitteri, S.J.; McLuckey, S.A. Parallel ion parking of protein mixtures. Anal. Chem. 2006, 78, 310–316. 98. Chrisman, P.A.; Pitteri, S.J.; McLuckey, S.A. Parallel ion parking: Improving conversion of parents to irst-generation products in electron transfer dissociation. Anal. Chem. 2005, 77, 3411–3414. 99. Goeringer, D.E.; Asano, K.G.; McLuckey, S.A.; Hoekman, D.; Stiller, S.W. Filtered noise ield signals for mass-selective accumulation of externally formed ions in a quadrupole ion-trap. Anal. Chem. 1994, 66, 313–318. 100. Grosshans, P.B.; Ostrander, C.M.; Walla, C.A. Methods and apparatus to control charge neutralization reactions in ion traps. U.S. Patent 2003, 6,570,151. 101. Grosshans, P.B.; Ostrander, C.M.; Walla, C.A. Methods and apparatus to control charge neutralization reactions in ion traps. U.S. Patent 2004, 6,674,067. 102. McLuckey, S.A.; Reid, G.E.; Wells, J.M. Ion parking during ion/ion reactions in electrodynamic ion traps. Anal. Chem. 2002, 74, 336–346. 103. Schwartz, J.C.; Syka, J.E.P.; Jardine, I. High-resolution on a quadrupole ion trap massspectrometer. J. Am. Soc. Mass Spectrom. 1991, 2, 198–204. 104. Stephenson, J.L.; McLuckey, S.A. Ion/ion proton transfer reactions for protein mixture analysis. Anal. Chem. 1996, 68, 4026–4032. 105. McLuckey, S.A.; Stephenson, J.L.; Goeringer, D.E. Gas-phase bio-ion/ion reactions: Charge transfer and ion pairing. Abstr. Pap. Am. Chem. Soc. 1996, 212, 17.I 106. Phanstiel, D.; Brumbaugh, J.; Berggren, W.T.; Conard, K.; Feng, X.; Levenstein, M.E.; McAlister, G.C.; Thomson, J.A.; Coon, J.J. Mass spectrometry identiies and quantiies 74 unique histone H4 isoforms in differentiating human embryonic stem cells. Proc. Natl. Acad. Sci. 2008, 105, 4093–4098. 107. Bowers, J.J.; Liu, J.; Gunawardena, H.P.; McLuckey, S.A. Protein identiication via iontrap collision-induced dissociation and examination of low-mass product ions. J. Mass Spectrom. 2008, 43, 23–34. 108. Newton, K.A.; Amunugama, R.; McLuckey, S.A. Gas-phase ion/ion reactions of multiply protonated polypeptides with metal containing anions. J. Phys. Chem. A 2005, 109, 3608–3616. 109. Newton, K.A.; McLuckey, S.A. Generation and manipulation of sodium cationized peptides in the gas phase. J. Am. Soc. Mass Spectrom. 2004, 15 (4), 607–615. 110. He, M.; Emory, J.F.; McLuckey, S.A. Reagent anions for charge inversion of polypeptide/protein cations in the gas phase. Anal. Chem. 2005, 77, 3173–3182.

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111. He, M.; McLuckey, S.A. Increasing the negative charge of a macroanion in the gas phase via sequential charge inversion reactions. Anal. Chem. 2004, 76, 4189–4192. 112. He, M.; McLuckey, S.A. Two ion/ion charge inversion steps to form a doubly protonated peptide from a singly protonated peptide in the gas phase. J. Am. Chem. Soc. 2003, 125, 7756–7757. 113. Han, H.L.; Xia, Y.; McLuckey, S.A. Ion trap collisional activation of c and z(center dot) ions formed via gas-phase ion/ion electron-transfer dissociation. J. Proteome Res. 2007, 6, 3062–3069.

Part II Ion Conformation and Structure

Derivatization 4 Chemical and Multistage Tandem Mass Spectrometry for Protein Structural Characterization Jennifer M. Froelich, Yali Lu, and Gavin E. Reid CONTENTS 4.1 4.2

4.3

4.4

Introduction ....................................................................................................84 Protein Identiication and Characterization .................................................... 85 4.2.1 Analytical Strategies to Overcome the Mixture Complexity and Dynamic Range Limitations Associated with Proteome Analysis .............................................................................. 85 4.2.1.1 Protein and Peptide Separation ............................................ 85 4.2.1.2 Protein and Peptide Depletion ............................................. 85 4.2.1.3 Protein and Peptide Enrichment and ‘Targeted’ Analysis................................................................................ 86 4.2.2 Challenges Associated with the Application of Tandem Mass Spectrometry Strategies for Peptide Sequence Analysis and Characterization.................................................................................. 87 4.2.2.1 Chemical Derivatization Strategies to Direct the Fragmentation Reactions of Protonated Peptides Toward the Formation of ‘Sequence’ Product Ions ............. 88 4.2.2.2 Chemical Derivatization Strategies to Direct the Fragmentation Reactions of Protonated Peptides Toward the Formation of Diagnostic ‘Non-Sequence’ Product Ions ......................................................................... 91 Quantitative Analysis of Protein Expression .................................................. 93 4.3.1 Two-Dimensional Differential Gel Electrophoresis ........................... 93 4.3.2 Label-Free Quantitative Analysis .......................................................94 4.3.3 Stable-Isotope Labeling Quantitative Analysis ..................................94 Protein Structure, Protein Folding, and Protein–Protein Interactions ......... 101 4.4.1 Cross-linking Strategies Employing Afinity Tags or Stable Isotope Labels ................................................................................... 102 83

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4.4.2

Cross-linking Strategies Employing ‘Solution-phase’ Cleavage Sites ................................................................................... 104 4.4.3 Cross-linking Strategies Employing ‘Gas-phase’ Cleavage Sites .................................................................................. 104 4.5 Concluding Remarks .................................................................................... 109 References .............................................................................................................. 109

4.1

INTRODUCTION

Major goals within the ield of proteomics are to identify, to characterize, and to quantify changes in protein expression, as well as to characterize protein–protein interactions, either at a particular time throughout the cell cycle or in response to a particular type of stimulation (for example, disease). The outcome of this research should enable a more complete understanding of the processes which control normal cellular function and the changes in cell regulation that lead to the onset and progression of disease. Due to its speed, sensitivity, and speciicity, mass spectrometry (MS) has become one of the leading technologies in the ield of proteomics [1]. The development of soft ionization techniques such as electrospray ionization (ESI) [2] and matrix-assisted laser desorption ionization (MALDI) [3], which have enabled large biological molecules to be ionized with minimal fragmentation, as well as signiicant advances in the bioinformatics tools which are employed for data analysis [4–7], have contributed to the overall success of MS-based proteomics research. Although a variety of instrumentation platforms are currently available, the quadrupole ion trap mass spectrometer offers the signiicant advantage of being able to perform multistage tandem mass spectrometry (MS n) to obtain detailed structural information for an ion of interest. Thus, aside from their relatively low cost and high sensitivity, the MS n capabilities of the quadrupole ion trap make this type of mass spectrometer particularly well-suited for the identiication and structural characterization of biological molecules. The ‘bottom-up’ or ‘shotgun’ tandem mass spectrometric (MS/MS) approach has emerged as one of the dominant methods employed for protein identiication, characterization, and quantitative analysis and for the characterization of protein–protein interactions. A typical bottom-up approach involves the enzymatic digestion of either unresolved protein mixtures, or individual proteins that have been resolved by electrophoretic or chromatographic methods. The resultant peptide mixture is fractionated using one or two-dimensional capillary liquid chromatography (LC) and introduced to the mass spectrometer via ESI or MALDI [8,9]. To obtain detailed structural information regarding the amino acid sequence of the peptide, or to identify and to localize modiication sites to particular amino acid residues located within the peptide sequence, individual protonated precursor ions are isolated automatically and subjected to MS/MS analysis [10,11]. The identiication of each peptide, and the protein from which it was derived, is achieved subsequently by de novo sequencing [10] or by database search algorithms which correlate the experimental product ion mass spectra with

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product ion mass spectra generated theoretically for peptides of the same mass contained within a known protein sequence database [4–7]. While the bottom-up approach has proven successful, there are several drawbacks, which limit its comprehensive application. This chapter will provide an overview of strategies that have been developed to address these limitations, with particular emphasis on the role of chemical derivatization strategies for enhancing the capabilities of multistage tandem mass spectrometric methods for targeted protein identiication, characterization, and quantitative analysis, and for the characterization of protein–protein interactions.

4.2 4.2.1

PROTEIN IDENTIFICATION AND CHARACTERIZATION ANALYTICAL STRATEGIES TO OVERCOME THE MIXTURE COMPLEXITY AND DYNAMIC RANGE LIMITATIONS ASSOCIATED WITH PROTEOME ANALYSIS

The increase in sample mixture complexity resulting from proteolytic digestion, and the dynamic range associated with the proteome, present formidable challenges for protein identiication and characterization. To address these challenges, a range of analytical strategies, involving the extensive separation, depletion, enrichment, or ‘targeted’ analysis of proteins, or their proteolytically-derived peptides, have been developed recently. 4.2.1.1 Protein and Peptide Separation Off or on-line one or two-dimensional chromatography methods are employed routinely to fractionate extensively intact protein mixtures [12], or proteolyticallyderived peptide mixtures originating from puriied proteins [13], prior to their analysis by MS. Numerous other strategies have been used in conjunction with chromatographic separation to increase the number of unique peptide ions selected for analysis by MS/MS, particularly for those present at low abundance. Dynamic exclusion is one such approach, whereby the m/z-values of precursor ions selected previously for fragmentation are placed automatically into an exclusion list for a deined period of time to prevent their re-selection [14–16]. An iterative survey scan approach has been described also, which subjects peptide mixtures to multiple replicate LC-MS/MS analyses [14–16]. In each individual analysis, precursor ions are selected for fragmentation from a narrow m/z-window rather than from the entire m/z-range. In addition, Wang and Li have described recently a strategy by which the m/z-values of peptide ions that are identiied positively in an initial LC-MS/MS run are placed into an exclusion list in subsequent runs of the same peptide mixture to prevent these peptides from being re-selected for fragmentation [17]. 4.2.1.2 Protein and Peptide Depletion The identiication of low abundance proteins present in complex biological samples (for example, human serum or cerebrospinal luid) can be improved also by removing

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proteins which are present at high abundance. Traditionally, the removal of such proteins has been achieved using dye–ligand afinity chromatography [18] or antibody-based methods such as immunoafinity chromatography [19,20]. In an alternative approach, combinatorial ligand library beads have been employed recently to increase the concentration of low abundance proteins while effectively reducing the concentration of high abundance proteins [21–23]. In this approach, a protein mixture is exposed to beads which have been synthesized with a library of diverse ligands. Each individual protein or peptide present within the sample will bind to the ligand exhibiting the strongest intermolecular interaction. Those proteins that are present at high abundance will continue to bind to the beads until a saturation limit is reached. However, low abundance proteins, which are present at concentrations below the saturation limits of the beads, will be bound extensively. Using this approach, the dynamic range of protein concentrations present within a complex biological sample can be reduced signiicantly. 4.2.1.3 Protein and Peptide Enrichment and ‘Targeted’ Analysis In efforts to decrease sample mixture complexity and to improve dynamic range, numerous ‘targeted’ approaches have been described, which analyze only a subset of the peptides contained within a proteolytically-derived peptide mixture. For example, afinity capture methods have been employed extensively for the enrichment of peptides containing speciic post-translational modiications or selected amino acid residues. Immobilized metal-ion afinity chromatography (IMAC) incorporating Fe3 + , Ga3 + , or Al3 + , has been used to isolate phosphorylated peptides [24,25], while the enrichment of histidine-containing peptides has been achieved using IMAC columns loaded with Cu2 + [26]. In an analogous approach, organomercurial agarose beads have been employed to isolate cysteine-containing peptides from a tryptic digest of yeast cell lysates [27]. Metal–oxide afinity chromatography (MOAC) methods utilizing titanium dioxide (TiO2), zirconium dioxide (ZrO2), and aluminum oxide (Al2O3), have been used also for the highly selective enrichment of phosphopeptides as an alternative strategy to IMAC [28–30]. The enrichment of glycosylated peptides prior to mass spectrometric analysis has been achieved using lectin afinity chromatography [31]. As an alternative ‘targeted’ approach, peptide subsets may be enriched via the chemical derivatization of speciic functional groups within a peptide (that is, amino acid side chains) followed by isolation using afinity capture, covalent capture, or chromatographic strategies [14,32–48]. Speciic examples include the biotinylation of cysteine residues within a peptide and subsequent enrichment using streptavidinafinity chromatography [14,32,33], or the introduction of a quaternary amine tag to the side chain of cysteine residues followed by their isolation using strong cation exchange (SCX) chromatography [38]. Thiol-speciic covalent resins have been employed to enrich for cysteine-containing peptides [41–44]. For example, Wang et al. have used a cysteine covalent capture strategy to characterize the mouse brain proteome [44]. It was reported that, in conjunction with global tryptic digestion, a total of 7792 proteins were identiied following LC-MS/MS analysis using a linear quadrupole ion trap with 1564 proteins identiied exclusively from the sample which had been subjected to cysteine peptide enrichment.

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4.2.2

87

CHALLENGES ASSOCIATED WITH THE APPLICATION OF TANDEM MASS SPECTROMETRY STRATEGIES FOR PEPTIDE SEQUENCE ANALYSIS AND CHARACTERIZATION

Another challenge facing the identiication and characterization of protonated peptide ions is the ability to form a series of b- and y-type ‘sequence’ product ions, via fragmentation of the peptide amide bonds during low-energy collision-induced dissociation (CID) tandem mass spectrometry that are required typically for subsequent database analysis and protein identiication. It is well documented that the fragmentation reactions of protonated peptide ions are inluenced strongly by both the amino acid composition and charge state of the precursor ion (that is, proton mobility) [49]. For example, the formation of dominant b- or y-type sequence ions resulting from enhanced cleavage at the amide bond N-terminal to proline residues is observed frequently under ‘mobile’ proton conditions (that is, when the total number of ionizing protons exceeds the combined number of arginine, lysine, and histidine residues) [50]. Under ‘non-mobile’ proton conditions, where ionizing protons are ‘sequestered’ at the side chains of basic amino acids (for example, arginine, lysine, or histidine), the formation of dominant b- or y-type sequence ions resulting from enhanced fragmentation at the amide bond C-terminal to aspartic acid residues are observed commonly [51]. Under certain conditions of proton mobility, the presence of several common post-translational or process-induced protein modiications may result in the formation of dominant ‘non-sequence’ neutral-loss product ions, via fragmentations occurring at the modiied amino acid side chain [52]. Examples include the neutral loss of CH3SOH (64 Da) from methionine sulfoxide-containing peptides [53], the neutral loss of alkyl sulfenic acid (RSOH) from S-alkyl cysteine sulfoxide-containing peptides [54–57], the neutral loss of H3PO4 (98 Da) from phosphoserine or phosphothreoninecontaining peptides [58–61], the neutral loss of SO3 (80 Da) from O-sulfonoserine, O-sulfonothreonine, or thiosulfate(–S–SO3H) containing peptides [62–64], and the loss of a glycan moiety from O-linked N-acetylgalactosamine-containing peptides [65,66]. The formation of these non-sequence neutral-loss product ions in high relative abundance may limit the amount of sequence information that is available for unambiguous identiication of a peptide, or for localization of the modiication site to a particular amino acid residue within the peptide sequence. Despite this risk of losing sequence information, the observation of diagnostic non-sequence side chain neutral-loss product ions may also be beneicial, by enabling the identiication of these modiied peptides in the gas-phase, thereby reducing the sample mixture complexity. Furthermore, observation of these diagnostic non-sequence product ions could potentially improve the speciicity of database search analysis strategies by enabling searches against only a subset of the peptides contained within a protein sequence database. When required, additional sequence information can be obtained by automatically subjecting the initial neutral-loss product ion to MS3 analysis in the quadrupole ion trap [25,28,67–69]. Typically, however, only a fraction of the peptide precursor ions containing these modiications will give rise to the diagnostic product ion of interest at suficiently-high abundance to permit MS3 examination, due to the

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strong proton mobility dependence associated with the mechanisms responsible for these losses [53,57,61]. Thus, it may be desirable to direct the fragmentation reactions of protonated peptides toward the formation of diagnostic product ions, through the use of chemical derivatization approaches. 4.2.2.1 Chemical Derivatization Strategies to Direct the Fragmentation Reactions of Protonated Peptides Toward the Formation of ‘Sequence’ Product Ions In an effort to improve peptide identiication and characterization by de novo sequencing or database search algorithm strategies, numerous chemical derivatization approaches have been developed to direct the fragmentation reactions of peptides toward the formation of a series of b- and/or y-type sequence product ions. One such approach involves chemical derivatization of the side chains of basic amino acid residues within a peptide to alter proton afinity [70–73]. For example, the reagents acetylacetone [70] and malondialdehyde [71] have been employed to modify chemically guanidino groups on the side chain of arginine residues in an effort to decrease the proton afinity of these sites. By decreasing the proton afinity of the arginine side chain, an ionizing proton is less likely to be sequestered and is available, therefore, to initiate cleavage of the amide bonds along the peptide backbone. It has been demonstrated that quadrupole ion trap CID-MS/MS of these chemically-modiied arginine-containing peptides results in an increased number and intensity of band y-type sequence product ions compared to their non-derivatized counterparts, thereby improving peptide identiication [71]. In a similar approach, the ε-amino group of C-terminal lysine residues has been converted to an imidazole derivative via chemical modiication with 2-methoxy-4,5-dihydro-1H-imidazole [72,73]. In contrast to producing both b- and y-type ions, the increased proton afinity resulting from this chemical modiication results predominantly in the formation of a series of y-type ions, yielding a simpliied CID-product ion mass spectrum for interpretation, particularly for de novo peptide sequencing. The chemical derivatization of peptide N- or C-termini to incorporate a ixed positive or negative charge is yet another approach that has been employed extensively to direct peptide ion fragmentation toward the formation of a desired series of sequence product ions [74–84]. For example, it has been shown that the chemical derivatization of peptide N-termini with S-pentaluorophenyl [N-tris(2,4,6-trimethoxyphenyl) phosphonium]acetate bromide (TMPP-AcSC6F5 bromide) [75,76] (Scheme 4.1) or with [tris(2,4,6-trimethoxyphenyl)phosphonium]acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) [77] to form [tris(2,4,6-trimethoxyphenyl)-phosphonium] acetyl (TMPP-Ac) derivatives directs the gas-phase CID fragmentation reactions of protonated peptides toward the formation of a series of a- or b-type sequence product ions. A representative example is shown in Figure 4.1a and b for the doublycharged ([M + 2H]2 + ) underivatized and doubly-charged ([M + + H]2 + ) TMPP-Ac derivatized precursor ions of the tryptic peptide PHPFHFFVYK, which has been subjected to low-energy CID-MS/MS in a quadrupole ion trap [77]. In contrast to the underivatized peptide, N-terminal derivatization to form the quaternary phosphonium ion results predominantly in the formation of a b-type ion series, and, to a lesser extent, a-type ions. Adamczyk et al. have demonstrated that N-terminal b- and

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89

OCH3 +

H3CO

–

P–CH2–CO–SC6F5Br + NH2–Peptide–COOH OCH3

3

DMAP, 15´ Room temperature

OCH3 +

H3CO

P–CH2–CO–NH–Peptide–COOH OCH3

3

(TMPP-Ac-Peptide)

SCHEME 4.1 Reaction of S-pentaluorophenyl [N-tris(2,4,6-trimethoxyphenyl)phosphonium] acetate bromide (TMPP-AcSC6F5 bromide) with the N-terminus of a peptide to form the [tris(2,4,6-trimethoxyphenyl)phosphonium]acetyl (TMPP-Ac) derivative. (Reproduced from Sadagopan, N.; Watson, J.T., J. Am. Soc. Mass Spectrom. 2000, 11, 107–119. With permission from the American Society for Mass Spectrometry, published by Elsevier Inc.)

a-type ions are formed predominately following CID-MS3 analysis of the TMPP-Ac containing b-type ions produced in the irst stage of tandem mass spectrometry analysis [77], which can be particularly useful for the analysis of larger peptides, for which incomplete sequence information is obtained frequently following CID-MS/ MS alone. Recently, ixed-charge chemical derivatization of peptide N-termini with (N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosponium acetate to form an acetylphosphonium derivative has been used to increase the sequence coverage obtained by electron capture dissociation (ECD) of o-phosphorylated and o-glycosylated peptides [79]. Peptide N-termini have been modiied chemically also with 4-sulfophenyl isothiocyanate and various other sulfonic acid derivatives to incorporate a ixed negative charge [81–84]. The introduction of a ixed negative charge on the N-terminus of the peptide, and the localization of an ionizing proton on the side chain of arginine or lysine residues contained within the peptide sequence, results in the formation of a neutral peptide molecule. To analyze the peptide via MS, a second ionizing proton is, therefore, required. Because the arginine or lysine side chains are already protonated, the second ionizing proton is able to move along the peptide backbone and initiate cleavage of the amide bonds. Using this approach, a single series of y-type sequence product ions are generated. Numerous chemical derivatization strategies have been developed also to direct the fragmentation reactions of protonated peptides toward the selective formation of single characteristic sequence-type product ions [85–87]. For example, Summerield and coworkers have demonstrated that N-terminal derivatization, with phenylisothiocyanate to form the corresponding phenylthiocarbamoyl (PTC) derivative, results in exclusive fragmentation of the amide bond between the irst two amino

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V (a)

y8

100

60

40

y82+ b82+ y4

y2 0 200

400

(b)

y5

600

PFHFVY

20

PFHFFV

b2 [y9–17]2+

Relative abundance

80

y6 b7 y7 b 8

800 m/z

1000

1200

1400

b82+

100

b72+

0 500

a7

b5

a6 2+

20

2+

700

[b9+18]2+

2+

b62+

b2 b92+ a92+

40

a82+

60

a52+

Relative abundance

80

900

b4 a4

b5

1000

b6 1300

b7

b8

1500

1700

1900

m/z

FIGURE 4.1 Quadrupole ion trap CID-product ion mass spectra of (a) the doubly-charged, [M + 2H]2 + , tryptic peptide PHPFHFFVYK (m/z 660), and (b) its doubly-charged, [M+ + H]2 + , [tris(2,4,6-trimethoxyphenyl)phosphonium]acetyl (TMPP-Ac) derivative (m/z 946). (Reproduced from Adamczyk, M.; Gebler, J.C.; Wu, J., Rapid Commun. Mass Spectrom. 1999, 13, 1413–1422. With permission from John Wiley & Sons.)

acid residues under mobile proton conditions to generate a b1 ion and the complementary yn–1 ion [85,86]. The incorporation of a ixed-charge TMPP-Ac tag to the N-terminus of aspartic acid-containing peptides has been employed also to promote enhanced cleavage C-terminal to aspartic acid residues [87]. The speciic information obtained, regarding the presence and location of an aspartic acid residue within a peptide sequence, has been shown to improve the speciicity of database search analysis strategies employed for protein identiication [88].

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4.2.2.2 Chemical Derivatization Strategies to Direct the Fragmentation Reactions of Protonated Peptides Toward the Formation of Diagnostic ‘Non-sequence’ Product Ions Recently, a ixed-charge chemical derivatization strategy, termed Selective Extraction of Labeled Entities by Charge derivatization and Tandem mass spectrometry (SELECT), has been developed to direct the fragmentation reactions of peptide ions toward the formation of diagnostic non-sequence product ions, so as to improve the capabilities of tandem mass spectrometry for selective peptide identiication and characterization [89–91]. In an initial demonstration of this approach, the side chains of methionine residues within a peptide or protein were alkylated with phenacylbromide (BrCH2COC6H5) to yield a ixed-charge sulfonium ion (Scheme 4.2) [89,90]. Under low-energy CID-MS/MS conditions, fragmentation of these sulfonium ioncontaining peptides was directed exclusively toward the site of the ixed-charge resulting in the formation of a single diagnostic product ion, independent of the proton mobility of the precursor ion. A representative example is shown in Figure 4.2 for a tryptic digest of yeast enolase, derivatized with a 1000-fold molar excess of phenacylbromide, subjected to ESI, and then examined by multistage tandem mass spectrometry (MS/MS and MS3) in a linear quadrupole ion trap. The full scan MS spectrum is shown in Figure 4.2a to demonstrate the mixture complexity associated with the sample. The CID product ion mass spectra obtained for the doubly- and triply-charged precursor ions, [M + + H]2 + and [M + + 2H]3 + , respectively, of the side chain phenacyl sulfonium ion ixed-charge derivative of the methionine-containing peptide AAQDSFAAGWGVMVSHR are shown in Figure 4.2b and c, respectively. The observation of a single diagnostic product ion corresponding to the neutral loss of phenacyl methyl sulide (CH3SCH2COC6H5, 166 Da) enables methioninecontaining peptides to be selectively ‘enriched’ in the gas-phase from within the complex proteolytically-derived peptide mixture. To obtain further structural information regarding the amino acid sequence of the peptide, the initial neutral loss product ions from Figure 4.2b and c were then isolated and subjected to further dissociation via MS3 (Figure 4.2d and e).

H3C

O C

N H

H3C

S CH2

(i) BrCH2COC6H5

CH2

(ii) MS

CH

C O

[M]

H N

O C

+ S

C

CH2

O

CH2 N H

CH

C

H N

(iii) CID –CH3SCH2COC6H5

[M +nH –CH3SCH2COC6H5](n+1)+

O [M+nH+CH2COC6H5](n+1)+

SCHEME 4.2 Fixed-charge derivatization and selective gas-phase fragmentation of methionine ixed-charge sulfonium ion derivatized peptides. (Reproduced from Amunugama, M.; Roberts, K.D.; Reid, G.E., J. Am. Soc. Mass Spectrom. 2006, 17, 1631–1642. With permission from the American Society for Mass Spectrometry, published by Elsevier Inc.)

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(a) 369.1

% Relative abundance

100

553.0

A A Q D S FA A G W G V M V S H R

379.1 392.5 490.8

[M++2H]3+ 637.0 709.1

600

400

[M++H]2+ 955.5

815.7

800

(b)

m/z

1200

1400

(c) [M++H–CH3SCH2COC6H5]2+

% Relative abundance

100

[M++2H–CH3SCH2COC6H5]3+

100

[M++H–2H2O]2+ [M++H]2+ [M++2H]3+

400 600 800 1000 1200 1400 1600 1800 (d)

200

400

600

800

1000

(e)

100 % Relative abundance

1000

–H2O

y153+

100

y142+ y152+

y153+ y6 y7 y2

y3 y4

y5

y112+ 2+ y12 2+ y9 –H O y132+ 2

y13 y9 b14 b15 y y8 b 10 y11 b16 10 b12

400 600 800 1000 1200 1400 1600 1800 m/z

b3 y2 b4 b5

200

400

b6

b7 b8

600 m/z

b9

800

b10

1000

FIGURE 4.2 Linear quadrupole ion trap multistage tandem mass spectrometry (MS/MS and MS3) analysis of a reduced and S-carboxyamidomethylated tryptic digest of yeast enolase following sulfonium ion derivatization with phenacylbromide. The mass spectrum obtained by ESI is shown in panel (a). The CID product ion mass spectra of the [M +  + H]2 + (m/z 955.5) and [M +  + 2H]3 + (m/z 637.0) precursor ions of the methionine side chain phenacyl sulfonium ion ixed-charge derivative of AAQDSFAAGWGVMVSHR are shown in panels (b) and (c), respectively. Panels (d) and (e) show the CID-MS3 product ion mass spectra of the [M +  + H –CH3SCH 2COC6H5]2 + (m/z 872.5) and [M + + 2H–CH3SCH 2COC6H5]3 + (m/z 581.7) product ions, respectively. Key: * = –NH 3.

More recently, this strategy has been applied to the selective identiication and characterization of cysteine-containing peptides, through the introduction of a ixed-charge on the side chain of cysteine residues via chemical derivatization with the reagent (3-[N-bromoacetamido]propyl)-methylphenacylsulfonium bromide (BAPMPS) [92]. Regardless of the amino acid targeted, the SELECT approach addresses issues associated with the identiication and characterization of

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protonated peptides by reducing the sample mixture complexity in the gas-phase, thereby improving the dynamic range. Furthermore, this approach has the potential to improve the speciicity of current database search analysis strategies, by enabling searches against only a subset of the peptides contained within a protein sequence database (that is, those containing methionine or cysteine). A number of approaches described in the literature have demonstrated also that selective dissociation can be achieved by irst generating a radical site within a peptide or protein [93–96]. For example, Ly and Julien described recently an approach whereby reactive tyrosine residues within individual proteins were converted to 3-iodotyrosine under natively-folded conditions [95]. The modiied tyrosinecontaining proteins were ionized via ESI, introduced to a linear quadrupole ion trap and, subsequently, subjected to UV photodissociation which resulted in the formation of a radical site on the aromatic ring of the modiied tyrosine residues. Following re-isolation and low-energy CID, radical-directed selective cleavage adjacent to the tyrosine residues was observed, resulting in the formation of a-type sequence product ions. Similar results were obtained also for proteins containing exposed histidine residues, which were shown also to be susceptible to iodination. The information generated via this site-speciic fragmentation could be used potentially to reduce the computational time associated with database search analysis strategies.

4.3

QUANTITATIVE ANALYSIS OF PROTEIN EXPRESSION

In addition to protein identiication and characterization, another major goal of proteomics research is to quantify protein expression levels. However, MS is not inherently quantitative. Thus, the intensity of a peptide ion introduced to the mass spectrometer via ESI or MALDI does not relect necessarily the amount of peptide present in the sample, due to the strong dependence of ionization on the physical and chemical nature of the analyte. To overcome this challenge, numerous quantitative analysis strategies have been developed to measure the differences in protein abundances between two different cellular states of a biological system (for example, normal and diseased cells).

4.3.1

TWO-DIMENSIONAL DIFFERENTIAL GEL ELECTROPHORESIS

Differential quantitative analysis has been performed previously at the protein level using two-dimensional differential gel electrophoresis (2D DIGE) [97–99]. In this approach, individual protein populations are labeled covalently with structurally similar, but spectrally distinct, luorophores. The protein populations are then combined and separated by 2D polyacrylamide gel electrophoresis (PAGE). Protein quantitation is achieved via imaging of the gel using different luorescence excitation wavelengths. To determine the identity of those proteins whose cellular expression levels are either up or down-regulated, gel spots are individually excised, subjected to enzymatic in-gel digestion, and the resultant peptide mixture is analyzed subsequently by MS. Although this technique overcomes many of the disadvantages associated with protein quantitation via traditional 2D PAGE, it still suffers from limited dynamic range (104), as well as a limited ability to resolve proteins with extremes

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of molecular weight and pI (where pI is the pH at which a molecule carries zero net electrical charge). In addition, accurate quantitative analysis is precluded when two or more proteins are present in the same gel spot.

4.3.2

LABEL-FREE QUANTITATIVE ANALYSIS

The quantitative analysis of protein expression levels from different protein populations has been achieved also using ‘label-free’ MS-based approaches [100–103]. In the label-free approach, control and experimental samples are digested enzymatically and analyzed individually by LC-MS and MS/MS. Protein abundances are then determined by either summing the extracted ion chromatographic peak areas [100,103], by summing the peptide identiication scores obtained from database analysis [101], or by summing the MS/MS spectral counts [102,103], for all the peptide ions identiied from a single protein. However, in order to achieve accurate quantitation, highly-reproducible LC-MS analysis is required to minimize shifts in retention time and luctuations in ion signal intensity. Furthermore, protein quantitation may be precluded when either peptides are present at low abundance or isobaric peptide ions elute at the same retention time.

4.3.3

STABLE-ISOTOPE LABELING QUANTITATIVE ANALYSIS

Differential quantitative analysis has been achieved also via the incorporation of differential stable isotope labels between control and experimental samples. To date, the majority of stable isotope-labeling methods have involved either in vivo metabolic labeling [104–106] or in vitro chemical derivatization [32,33,35,41–43,106–108]. In vivo metabolic labeling approaches such as SILAC (Stable Isotope Labeling by Amino acids in Cell culture) [105] incorporate a differential stable isotope label by growing one population of cells in normal media and a second population of cells in media enriched with an isotopically-encoded amino acid. Following protein extraction, the two protein populations are combined and digested enzymatically. Protein expression levels are determined subsequently by MS analysis via comparison of the relative abundances of intact peptide precursor ions derived from the ‘light’ and ‘heavy’ isotopically-labeled samples. In vitro chemical derivatization approaches employed for differential quantitative analysis either label all peptides within a proteolytically-derived peptide mixture (that is, at the N- or C-terminus), or target speciic amino acid side chains or posttranslational modiications. A general overview of commonly-employed chemical derivatization strategies is shown in Figure 4.3. A more detailed discussion of each of these chemical derivatization approaches can be found in a review article by Julka and Regnier [106] and the references cited therein. Although numerous chemical derivatization and quantitative analysis strategies have now been described, one of the earliest of these involved use of the isotope-coded afinity tag (ICAT) reagent [32]. The irst generation ICAT reagent, designed by Gygi et al., consisted of an iodoacetyl thiol-speciic reactive group, a biotin tag, and an oxyethylene linker region which contained either eight hydrogen atoms (light ICAT reagent) or eight deuterium atoms (heavy ICAT reagent) [32]. In this approach, all cysteine residues from

(55) R–CHO

F

Biotin-SS

(17) NO2

SCI

N HN I RNHCOCH 2 OH SH CH3 CH2 CH2 H2NCHCONHCHCONHCHNHCH2CONHCH2CONHCHCONHCHCONHCHCONHCH2COOH CH2 CH2 CH2 COOH CH2 CH2 NH2 (68) (22,57,58) 18 CH3OH O Labeling (68)CH3OH (116) OMe (108,109,110) NH2 HN N CH3O NH2

N R

(88,89) (100) O CH2CHCONH2

FIGURE 4.3 Generic summary of the stable isotope labeling strategies employed currently for MS-based relative protein quantitation. All reactions shown to occur on the amino terminus apply also to the ε-amino group of lysine residues. The numbers in parentheses indicate the references cited in the review article by Julka and Regnier. (Reproduced from Julka, S.; Regnier, F., J. Proteome Res. 2004, 3, 350–363. With permission from the American Chemical Society.)

(51) O2N

(54) RNCS NO2

(13) RCOOOCR

(11,14,18) R–CO–OR

O (16,26,27) RNHCOCH2I

(102)

Chemical Derivatization and Multistage Tandem Mass Spectrometry 95

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control and experimental samples are labeled with the light and heavy ICAT reagent, respectively. After labeling, the two protein populations are combined, subjected to enzymatic digestion, and then the ICAT-labeled peptides are isolated from nonlabeled peptides using avidin afinity chromatography. Relative protein expression levels are then determined by measuring the peak ion signal intensity ratios of peptide pairs in a mass scan, while peptide identiication is achieved by MS/MS analysis of individual peptide precursor ions. Using the ICAT approach, protein quantitation and a reduction in sample mixture complexity are achieved simultaneously. Despite the initial success of this approach, a number of disadvantages associated with the irst generation ICAT reagent have been noted. For example, the presence of deuterium atoms in the linker region of the heavy ICAT reagent may result in the chromatographic separation of light and heavy ICAT-labeled peptides during reversed-phase chromatography, thereby precluding their accurate quantitation. To ensure that light and heavy ICAT-labeled peptides co-elute, a second generation ICAT reagent was designed to include 13C rather than 2H in the linker region [33]. In addition, fragmentation of the bulky biotin tag has been observed frequently during CID-tandem mass spectrometric analysis, which complicates interpretation of the resultant product ion mass spectra for peptide identiication. Thus, an acid-cleavable group, which connects the biotin moiety with the thiol-speciic isotope tag, was also incorporated into the second generation ICAT reagent to cleave the biotin moiety from modiied peptides [33]. In contrast to the irst generation ICAT reagent, the small size of the remaining tag results in minimal fragmentation following CIDtandem mass spectrometry. As an alternative to the solution-phase ICAT approach, a number of solid-phase isotope-labeling strategies have been developed [41–43]. Solid-phase covalent capture methods enable more stringent wash conditions to be employed in an effort to remove non-speciically bound peptides. In addition, peptide labeling and isolation can be achieved in a single step. Zhou et al. have described a method for solid-phase stableisotope labeling of cysteine-containing peptides from Saccharomyces cerevisiae using controlled-pore glass beads containing an o-nitrobenzyl-based photocleavable linker, a stable isotope tag incorporating either seven hydrogen or seven deuterium atoms, and a thiol-speciic iodoacetyl group [41]. Proteins from control and experimental samples were proteolyzed individually and then cysteine-containing peptides were captured by d0- or d7-beads, respectively. The beads were then combined, washed, and cleaved photolytically to release the differentially-labeled cysteine-containing peptides. Following LC-MS/MS analysis with a quadrupole ion trap, it was determined that more proteins could be identiied and quantiied using the solid-phase isotope-labeling approach than when the samples were prepared using the irst generation solution-phase ICAT reagent. A similar solid-phase isotope-labeling approach termed acid-labile isotope-coded extractants (ALICE) has also been developed by Qiu et al. [42]. In this approach, cysteine-containing peptides are captured using a non-biological polymer which has been modiied chemically to contain a maleimido thiol-reactive group and an acid-labile linker in both heavy and light isotope-encoded forms. For the majority of stable-isotope labeling approaches, including those described above, quantitative analysis is achieved in a mass scan. Limitations are encountered,

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however, when one or both of the differentially-labeled peptide ions are present at or below the level of chemical noise in the mass spectrum, thereby precluding quantitative analysis. Furthermore, accurate quantitation may be precluded when the m/z values of differentially-labeled peptide ions overlap with non-labeled or other labeled components present in the peptide mixture. To address these challenges, several differential quantitative analysis strategies have been described recently whereby quantitation is performed by tandem mass spectrometry, rather than by a mass scan [109–115]. The iTRAQ approach, which utilizes a commercially-available multiplexed set of reagents to quantitate relative expression levels for multiple protein populations, is one example [112,113]. The initial reagent employed in this approach consists of a reporter group, a balance group, and an amine speciic peptide reactive group (Figure 4.4a). Differential stable isotope labels are incorporated into the balance and reporter groups of four iTRAQ reagents in such a way that the tag generated upon reaction with a peptide has the same overall mass ( + 145.1 Da) (Figure 4.4b). To use this approach for quantitative analysis, peptide mixtures are labeled individually with one member of the multiplexed set after which the labeled peptide mixtures are combined and subjected to mass spectrometric analysis (Figure 4.4c). In a mass scan, identical tagged peptides from each of the four samples are present at the same m/z value, therefore the sensitivity in a mass scan is maximized. Upon CID-tandem mass spectrometric analysis of the peptide precursor ions, the balance group is lost as a neutral, while the reporter group retains a charge to generate low m/z product ions at m/z 114, 115, 116, or 117, which are used subsequently for quantitative analysis. The b- and y-type ions that may be generated also during the MS/MS experiment remain isobaric and can be used to identify the sequence of the labeled peptide. Compared to conventional MS-based approaches, increased sensitivity and greater speciicity is achieved due to the reduction in chemical noise associated with the MS/MS technique. To increase the number of protein populations, which can be analyzed via this approach, an 8-plex version of the iTRAQ strategy was introduced recently [114]. In a recent publication, Li and Zeng have described a similar tandem mass spectrometric-based quantitative analysis strategy termed Cleavable Isobaric Labeled Afinity Tag or CILAT [115]. Essentially a hybrid of the ICAT and iTRAQ approaches, the CILAT reagent includes an isobaric tag consisting of a reporter group and a balance group. The reagent incorporates also a biotin moiety for the enrichment of modiied peptides via avidin afinity chromatography and an acid cleavable linker to remove the biotin moiety prior to MS/MS analysis. The thiol group employed in this reagent is used to modify tyrosine residues within peptides that have been converted to ortho-quinone via oxidation with tyrosinase. Although quantitative measurement strategies based on tandem mass spectrometric analysis overcome the limitations associated with normal mass scanning approaches, the product ions of low m/z required for quantitation are often found to lie below the low mass cut-off introduced during MS/MS in quadrupole ion trap mass spectrometers, thereby precluding their use for quantitative analysis in this type of instrumentation. Thus, to date, the majority of proteomic experiments utilizing these reagents has been performed using quadrupole time-of-light (QTOF), timeof-light/time-of-light (TOF/TOF) and, to a lesser extent, hybrid triple-quadrupole/

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(a) Isobaric tag Total mass = 145

H N

Amine specific peptide reactive group (NHS)

Reporter group mass 114 – 117 (Retain charge)

N

O

Pepti d e O

N

O N N

N O O

Balance group Mass 31–28 (neutral loss)

m/z 114 (+1)

13

m/z 115 (+2)

13

m/z 116 (+3)

13

m/z 117 (+4)

13

13

C

C

C2

O (+3)

18

O (+2)

C2 C2

18

15

N

13

(+1)

C

15

(+0)

N

(c) 31

NHS + pe pt id e

115 30

NHS + pe pt id e

114

116

29

NHS + pe pt id e

114 114 31 -NH-pep tid e MS/MS 115 30 -NH-p ep tid e 116 29 -NH-p ep tid e MS 117 28 -NH-p ep tid e

Mix

b 115 P 116

E

P

T

I

D

E y

117 117

28

NHS + pe pt id e Reporter-Balance-Peptide INTACT –4 Samples identical m/z

-Peptide fragments EQUAL -Reporter ions DIFFERENT

FIGURE 4.4 (a) Structure of the iTRAQ reagent that consists of a reporter group, a mass balance group, and a peptide reactive group. The overall mass of the reporter and balance components of the molecule is kept constant (145.1 Da) using differential isotopic enrichment with 13C, 15N, and 18O atoms. (b) Upon reaction with a peptide, the tag forms an amide linkage to any peptide amine (N-terminus or ε-amino group of lysine). When subjected to CID, fragmentation of the tag amide bond results in the loss of the balance group as a neutral species, while the charge is retained by the reporter group fragment. The numbers in parentheses indicate the number of enriched centers in either the reporter group or balance group of the molecule. (c) A mixture of four identical peptides, each labeled with one member of the multiplex set, appears as a single, unresolved precursor ion in a mass scan (identical m/z). Following CID, the four reporter group ions appear as distinct masses (m/z 114–117). All other sequence-informative fragment ions (that is, b- and y-type ions) remain isobaric. (Reproduced from Ross, P.L.; Huang, Y.N.; Marchese, J.N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D.J., Mol. Cell. Proteomics. 2004, 3, 1154– 1169. With permission of the American Society for Biochemistry and Molecular Biology.)

linear ion trap mass spectrometers [116–120]. However, a new ‘High Amplitude Short Time Excitation’ (HASTE) dissociation technique (an analogous method is termed Pulsed Q Collision-Induced Dissociation (PQD) in the commercially-available ion trap mass spectrometer platforms available from Thermo Scientiic), has been implemented recently which enables the product ions of low m/z, excluded normally from product ion mass spectra, to be observed [121]. Using the iTRAQ approach coupled with PQD-MS/MS on a Thermo linear quadrupole ion trap, Meany et al. were able to quantify successfully carbonylated proteins enriched from rat skeletal

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muscle mitochondria [122]. The PQD-MS/MS product ion mass spectrum of the triply-protonated peptide IEGTPLEAMQKK, derived from the carbonylated protein NADH dehydrogenase 1 beta sub-complex 3, is shown in Figure 4.5a as a representative example [122]. An expansion of the low m/z region of the product ion mass spectrum depicted in Figure 4.5a shows the iTRAQ reporter ions at m/z 114–117 (a) 563.3

100 90 Relative abundance

80 70 60 291.3

50 40 30

617.4 447.9 674.4 527.3

894.6 823.6 750.5

145.0 116.1

20 10 0

200

400

600 m/z

(b) Control

100

1 115.1

2 116.1

90

800

1000

1200

3 117.1

80 70 60 50 40 30 20 10 0 113

114

115

116

117

118

119

FIGURE 4.5 (a) Linear quadrupole ion trap Pulsed Q Collision-Induced Dissociation (PQD)-tandem mass spectrometric analysis of the [M + 3H]3 + precursor ion of the peptide IEGTPLEAMQKK derived from the carbonylated protein NADH dehydrogenase (ubiquinone) 1 beta sub-complex 3. (b) An expansion of the low m/z region of the product ion mass spectrum in panel A shows the relative abundance of the iTRAQ reporter ions at m/z 114–117. The peptide showed increased abundance in the samples that had been labeled with biotin hydrazide prior to avidin enrichment (iTRAQ reporter ions m/z 115–117) as compared to the control sample that had not been labeled with biotin hydrazide prior to avidin puriication (iTRAQ reporter ion m/z 114). (Reproduced from Meany, D.L.; Xie, H.; Thompson, L.V.; Arriaga, E.A.; Grifin, T.J. Proteomics. 2007, 7, 1150–1163. With permission from Wiley-VCH Verlag GmbH.)

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which were used for quantitative analysis (Figure 4.5b). Grifin et al. also compared PQD-MS/MS in a linear quadupole ion trap mass spectrometer with MS/MS in a QTOF mass spectrometer for the quantitative analysis of iTRAQ-labeled peptides derived from a standard yeast lysate mixture [123]. It was determined that similar quantitative accuracy could be achieved using both instrumentation platforms, although optimization of the amplitude of the collision energy was required for eficient fragmentation by PQD. Protein kinases, extracted from cells that had been incubated with various drugs, have been quantiied also using the iTRAQ approach and PQD-MS/MS in a linear quadrupole-Orbitrap mass spectrometer [124]. It has been demonstrated also that the iTRAQ tandem mass spectrometric quantitative analysis strategy can be used in conjunction with the quadrupole ion trap by performing multiple stages of mass analysis (that is, MS3) [125]. For example, chemical derivatization with the iTRAQ reagent not only labels the N-terminus of a peptide, but the lysine side chain also. Thus, tryptic peptides with a modiied lysine residue present at the C-terminus will produce a y1 product ion at m/z 291 following CID-tandem mass spectrometry. To generate the low m/z iTRAQ reporter ions required for quantitation, the y1 product ion is isolated and subjected to datadependent CID-MS3. Using this approach, peptide identiication is achieved in the MS/MS scan, while quantitation is achieved via MS3. Regardless of the instrumentation platform employed for analysis, each of the quantitative tandem mass spectrometric derivatization strategies requires an ionizing proton to initiate cleavage of the stable isotope-containing label in order to generate the low m/z reporter ions required for quantitation. Therefore, the fragmentation reactions associated with these strategies are expected to be highly dependent upon the proton mobility of the precursor ion, such that the characteristic isotopically-encoded low mass reporter ions may often be observed at suficient levels in only a sub-set of the total peptide ions selected for MS/MS to enable their use for quantiication. Another limitation is that the desired fragmentation pathway giving rise to the low m/z reporter ions of interest is typically only one of many dissociation channels, including those resulting in the formation of b- and y-type sequence product ions, thereby ‘diluting’ the mass spectrum and limiting the dynamic range for quantitative analysis. To overcome some of these challenges, the SELECT approach described in Section 4.2.2.2 above has been applied to quantitative analysis, via the incorporation of light and heavy isotopically-encoded labels into the ixed-charge chemical derivatization reagents. In the initial report by Reid et al., methionine-containing peptides were alkylated with either ‘light’ 1H5-phenacylbromide or ‘heavy’ 2H5-phenacylbromide to form a ixed-charge sulfonium ion on the side chain of methionine residues [89]. The light and heavy phenacyl sulfonium ion ixed-charge-containing peptides were combined and subjected subsequently to LC-MS/MS analysis. The relative abundances of the neutral loss product ions generated by CID-MS/MS, formed via the loss of CH3SCH2COC6H5 (166 Da) and CH3SCH2COC62H5 (171 Da), respectively, were used for subsequent quantitative analysis. In contrast to the tandem mass spectrometric quantitative analysis strategies described above, the neutral loss product ions required for quantitation via the SELECT approach are formed independently of the proton mobility of the precursor ion, such that these product ions are observed under all experimental conditions. Furthermore, formation of the neutral loss product

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ions at a m/z value close to that of the selected precursor ion circumvents the lowmass cut-off limitation of the ion trap. Characterization of the identiied and quantiied methionine peptides may be achieved readily by subjecting the common neutral loss product ion formed from the light or heavy labeled peptides to MS3 analysis. However, one of the potential disadvantages to this initial approach is that some, albeit limited, chromatographic separation of the 1H5 and 2H5 forms of the ixed-charge sulfonium ion derivatives was observed following reversed-phase chromatography of these peptides. Thus, to ensure that the labeled peptides co-elute during reversedphase chromatographic separation, and to improve the capabilities of this approach, more recent studies have employed 12C6-phenacylbromide and 13C6-phenacylbromide derivatives for ixed-charge chemical derivatization of methionine-containing peptides [126]. Although not investigated to date, light and heavy isotopically-encoded labels could also be incorporated readily into the previously-described alkylating reagent (3-[N-bromoacetamido]propyl)-methylphenacylsulfonium bromide [92] to enable the selective tandem mass spectrometric quantitative analysis of cysteinecontaining peptides.

4.4

PROTEIN STRUCTURE, PROTEIN FOLDING, AND PROTEIN–PROTEIN INTERACTIONS

Due to their high sensitivity and rapid analysis capabilities, numerous mass spectrometric approaches have been developed also to study protein structures and the dynamics of protein folding as well as to characterize protein–protein interactions. For example, hydrogen–deuterium exchange combined with MS has been employed extensively and successfully to probe solvent-accessible regions of proteins and to examine protein folding [127]. A variety of covalent-labeling approaches have been used also to modify oxidatively the side chains of solvent-accessible amino acid residues followed by mass spectrometric analysis to determine the site(s) of modiication. Examples include the use of hydroxyl radical probes generated by (1) highenergy synchrotron radiolysis of water [128], (2) UV irradiation of hydrogen peroxide [129], (3) modiication using electrochemical oxidation [130], and (4) direct hydrogen peroxide oxidation [131]. Numerous chemical derivatization reagents including diethylpyrocarbonate, butanedione, and phenylboronic acid have been used also to modify speciic amino acid side chains [132–135]. Chemical cross-linking combined with MS is also a promising approach for studying the low-resolution structure of protein topology as well as for probing protein–protein interactions [136]. Cross-linkers can link covalently interacting regions either within a single protein or between individual subunits of multi-protein complexes. Most importantly, non-covalent protein–protein interactions, which may be transient or dependent on speciic physiological conditions, can be captured into long-lived covalent complexes [137]. In a typical bottom-up approach, cross-linked proteins or protein complexes are separated typically by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subjected subsequently to enzymatic in-gel digestion, chromatographic fractionation, and mass spectrometric analysis [138]. Ultimately, the assignment of distance constraints, within a single protein or protein

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complex, can be employed to provide information regarding the protein–protein interactions and three-dimensional structures of the proteins or protein complex. However, despite the relative simplicity of cross-linking approaches, the identiication of cross-linked products by MS is not straightforward due to the high complexity of the peptide mixtures generated via enzymatic digestion. The well-known term, “looking for a needle in a haystack” is used often to describe the challenges associated with the identiication of cross-linked peptides from within a large number of unmodiied peptides. In addition, numerous types of cross-linked peptides can be produced as a result of the chemical cross-linking reaction, which can complicate further peptide analysis. For example, dead-end modiied peptides (Type 0) are formed when one of the reactive groups of the cross-linker has reacted with a protein while the second reactive group has been hydrolyzed. A type 0 modiication does not provide any information related to the distance constraints between two amino acid residues, but may indicate reactive groups exposed on the protein surface. Intra-molecular cross-linked peptides (Type 1) are formed when both reactive groups of the cross-linker have reacted with two amino acid residues on the same peptide chain. Type 1 cross-linked peptides can be used to map the low-resolution three-dimensional structure of proteins. Finally, inter-molecular cross-linked peptides (Type 2) are formed when the reactive groups of the cross-linker have reacted with two amino acid residues on two different peptide chains. When the cross-linked peptides are from two proteins within a protein complex, information about the interacting sites can be obtained. To date, a number of chemical modiication techniques have been developed to address the challenges associated with the identiication of cross-linked peptides, including the enrichment of cross-linker-containing species by speciic afinity tags, the introduction of discriminating properties such as differential isotope labels, or by the introduction of speciic ‘solution-phase’ or ‘gas-phase’ cleavage sites, either within the cross-linking reagent itself or within the cross-linked peptides.

4.4.1

CROSS-LINKING STRATEGIES EMPLOYING AFFINITY TAGS OR STABLE ISOTOPE LABELS

Speciic afinity tags have been incorporated into the cross-linking reagent to enrich for cross-link-containing species prior to mass spectrometric analysis [139–143] (Figure 4.6a). Most commonly, a biotin functional group is incorporated into the cross-linker, followed by afinity puriication of the cross-linked peptides using either an avidin afinity column or avidin beads. Differential stable isotope labeling strategies have been employed also to detect cross-linked peptides [144–151]. Using this approach, isotopic labels are incorporated within the protein or peptide, or within the cross-linker itself, to produce a distinctive mass shift or isotopic pattern following mass spectrometric analysis (Figure 4.6b). One approach which incorporates a stable isotope label within the polypeptide chain involves the introduction of two 18O atoms to each of the C-terminal carboxyl groups during proteolytic digestion in 18O enriched water [145]. Thus, inter-molecular cross-linked peptides are distinguished readily by a characteristic mass shift of 8 Da compared with peptides formed upon proteolysis in natural abundance water. However, it is not possible to distinguish

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(b)

(a) Affinity labeled cross-linkers S

HN O

Stable isotope labeled cross-linkers

N H

“Light” “Heavy”

(c) Solution cleavable cross-linkers S

“Light”

S

“Heavy”

FIGURE 4.6 General representation of (a) afinity labeled, (b) stable isotope labeled, and (c) solution cleavable cross-linking strategies used to identify cross-linked peptides.

between dead-end cross-linked peptides, intra-molecular cross-linked peptides, and unmodiied peptides, as they will all exhibit the same mass increment of 4 Da following the introduction of two 18O atoms to their C-terminal carboxyl groups during proteolytic digestion in 18O enriched water. Another approach, described by Chen et al., involves reductive di-methylation of primary amino groups within a protein, followed by enzymatic hydrolysis and derivatization of the newly-formed N-termini with a 1:1 (w/w) mixture of 2,4-dinitroluorobenzene-d 0 /d3 [146]. Due to the incorporation of two dinitrophenyl groups, inter-molecular cross-linked peptides are distinguished by a characteristic 1:2:1 isotope pattern in the mass spectra from the 1:1 isotope pattern of other cross-linked types and non-modiied peptides. Also, for visualizing inter-molecular cross-linked peptides, a mixed isotope cross-linking (MIX) strategy was designed by mixing 1:1 uniformly 15N-labeled and unlabeled proteins to form a mixture of homodimers [147]. Molecular ions formed from crosslinked peptides of intermolecular origin are observed as a triplet or quadruplet of labeled peaks containing [15N/15N]/[15N/14N]/[14N/15N]/[14N/14N], while all other peptide species are observed as a doublet of [15N]/[14N] labeled peaks. While the introduction of stable isotope labels on either the protein or the peptide enables the ready identiication of inter-molecular cross-linked peptides, intramolecular cross-linked peptides cannot be distinguished by this method. Thus, isotopic labels have been incorporated directly into the cross-linking reagent. It has been demonstrated that by reacting with 1:1 (w/w) mixtures of stable isotope-labeled and non-labeled cross-linking reagents, cross-linked peptides can be detected readily by their distinctive 1:1 isotope pattern [148,149]. Isotope-labeled cross-linkers have been combined with other strategies for further identiication of cross-link types [150,151].

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4.4.2

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

CROSS-LINKING STRATEGIES EMPLOYING ‘SOLUTION-PHASE’ CLEAVAGE SITES

Although isotope-labeling technologies have been employed widely to assist in the identiication of cross-linked peptides, this approach still suffers from a number of limitations. For example, in a mass spectrum of high complexity, the characteristic isotopic pattern of cross-linked peptides will be masked either because of the small mass difference between isotopes, or because of the low relative abundances of the cross-linked products. Moreover, the use of only one isotope labeling process is not capable of identifying all the cross-linked types at once. Therefore, to improve the ability to identify and to sequence cross-linked peptides, a range of cleavable cross-linking reagents has been developed. The cleavage reaction can be performed in solution, by hydrolysis [151], or by the use of reducing agents in the case of disulide containing cross-linking reagents [152], (Figure 4.6c). The thiol-cleavable cross-linking reagent 3,3′-dithio-bis(succinimidylpropionate) (DTSSP) has been applied to a number of proteins and protein complexes [152–154]. After reduction of the cross-linker disulide bond, intermolecular cross-linked peptides give rise to two separated components, each containing a reduced linker. Intra-molecular cross-links yield two reduced-linker halves on the same peptide, whereas dead-end types only have one present. Different cross-link types are distinguished by their characteristic mass shifts before and after reduction. Another example involves cleavable cross-linkers combined with isotope-coding strategies [151]. The isotopically-labeled cleavable cross-linker is applied with its unlabeled counterpart to provide additional discriminating information because of the distinctive isotope-pairs thus, formed, allowing unambiguous identiication of cross-linked products. However, the application of cleavable cross-linkers by a chemical reaction relies on mass spectrometric analysis before and after the cleavage reaction takes place. Thus, the identiication of cross-linked products might be complicated when the corresponding signals of reduced cross-link species either are not observed, or they overlap with non-cross-linked peptides.

4.4.3

CROSS-LINKING STRATEGIES EMPLOYING ‘GAS-PHASE’ CLEAVAGE SITES

To simplify the problem, tandem mass spectrometric methods employing low energy CID have been applied recently to the cleavage and identiication of crosslink containing peptides in the gas-phase. The cleavage site can be incorporated on the side chain of the cross-linking reagent, resulting in the formation of a characteristic stable product ion or neutral loss upon MS/MS, while maintaining the cross-linked peptide linkage (Figure 4.7a). For example, Back et al. have described the use of a bifunctional lysine reactive cross-linker, N-benzyliminodiacetoylhydroxysuccinimid (BID), which yields a stable benzyl cation under low energy CID conditions, to identify successfully inter- and intra-molecular cross-linked peptides [155]. However, the low m/z-ratio of the benzyl cation can limit the application of BID when quadrupole ion trap instruments are used due to the low mass cut-off inherent to this instrument. In addition, the benzyl cation is observed only as a dominant product from certain precursor ion charge states of the protonated peptide. Bruce and co-workers have introduced a novel cross-linker strategy, termed protein

Chemical Derivatization and Multistage Tandem Mass Spectrometry Gas-phase cleavable cross-linkers

MS/MS

(a)

105

m/z

MS/MS

(b)

m/z

FIGURE 4.7 Tandem mass spectrometric strategies for the targeted identiication of crosslinked peptides. Gas-phase cleavable cross-linking reagents can be designed such that the cleavage site is incorporated either (a) into the side chain of the cross-linking reagent or (b) directly into the cross-linker spacer chain.

interaction reporter (PIR), which incorporates an afinity tag, a hydrophilic group, a photocleavable group, and low-energy CID-cleavable bonds [143,156]. Upon tandem mass spectrometry, PIR intermolecular cross-linked peptides fragment at two cleavage sites within the linker, giving rise to a reporter ion and two separated peptides each with an additional ixed mass. However, the spacer-arm chain length of nearly 43 Å for the PIR makes the use of this reagent less informative in determining the speciic interaction distances for protein–protein interactions. Alternatively, the cleavage site may be incorporated directly into the cross-linker spacer chain, resulting in cleavage of the cross-link upon MS/MS (Figure 4.7b). The incorporation of a single gas-phase cleavable bond within the linker region enables the use of MSn to determine the amino acid sequence of each peptide following the initial cleavage reaction. For example, Soderblom and Goshe have developed recently a set of single site gas-phase cleavable cross-linking reagents that can be fragmented selectively in the source region of the mass spectrometer (Figure 4.8) [157]. In this approach, the acquisition of a full mass scan is followed by in-source collision-induced dissociation (ISCID) due to the application of an additional potential offset between the skimmer lens and the multipole region of the mass spectrometer. Utilization of ISCID results in cleavage at the aspartyl-prolyl bond within the cross-linking reagent, which is possibly mediated by proton transfer from the aspartyl side chain to the basic amine of the adjacent prolyl residue. Thus, the ISCID mass spectral scan results in the formation of two peptide product ions, each containing a unique modiication corresponding to the remaining portion of the cross-linking reagent. The precursor ions

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V SuDP Bisuccinimidyl-succinamyl-aspartyl-proline OH

O O

O O

N

O N

N O

H

O O

O

O

O

N

11.2 Å SuDPG Bisuccinimidyl-succinamyl-aspartyl-prolyl-glycine O O N

O O

O

O N

N O

O

OH

H

O

O N

N H

O

O

15.1 Å

FIGURE 4.8 Structures of the collision-induced dissociative cross-linking reagents bisuccinimidyl-succinamyl-aspartyl-proline (SuDP) and bisuccinimidyl-succinamyl-aspartylproline-glycine (SuDPG). Each reagent incorporates a single gas-phase cleavable bond within the linker region for selective fragmentation in the source region of the mass spectrometer. The calculated distance between each of the reactive sites is indicated for each reagent. (Reproduced from Soderblom, E.J.; Goshe, M.B., Anal. Chem. 2006, 78, 8059–8068. With permission from the American Chemical Society.)

from the ISCID mass spectral scan are subjected subsequently to tandem mass spectrometry in order to identify the peptides and to localize the modiication introduced by the cross-linking reagent to speciic lysine residues within the peptide sequence. Regardless of whether the cleavage site is incorporated on the cross-linker side chain or in the cross-linker spacer chain, it would be desirable for selective fragmentation at these sites to occur prior to cleavage along the peptide backbone. However, the mechanisms responsible for the gas-phase fragmentation reactions that give rise to the product ions of interest within protonated peptide ions (including cross-linked protonated peptide ions) are dependent typically on both the charge state and the amino acid composition of the peptide (that is, proton mobility) [49]. Thus the characteristic product ions required for cross-link identiication may be observed only from a subset of the total cross-linked peptide ions that are subjected to dissociation. To improve the identiication of cross-linked peptides, and to develop a ‘targeted’ multistage tandem mass spectrometric-based approach for the identiication and characterization of protein–protein interactions, a ixed-charge sulfonium ioncontaining amine reactive cross-linking reagent, S-methyl 5,5′-thiodipentanoylhydroxysuccinimide, was synthesized recently, characterized and applied initially to the

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analysis of the cross-linked products formed by reaction with various model peptides (Scheme 4.3) [158]. Under low-energy CID conditions, peptide ions containing this cross-linker undergo fragmentation exclusively at the C–S bond adjacent to the ixedcharge, independent of the charge state and amino acid composition of the precursor ion. A representative example of this cross-linking strategy is shown in Figure 4.9 for the homo-dimer of cross-linked neurotensin (pELYENKPRRPYIL) subjected to multistage tandem mass spectrometric analysis (MS/MS and MS3) in a quadrupole ion trap. The full scan mass spectrum obtained following the cross-linking reaction is shown in Figure 4.9a; the m/z-value is indicated for the homo-dimeric intermolecular cross-linked peptide ([2M + 4H + (I–S)]5 + ) (where (I–S) indicates the presence of the cross-link). CID of the [2M + 4H + (I–S)]5 + precursor ion resulted in the formation of two characteristic product ions, ([M + 2H + I]3 + and [M + 2H + S]2 + ), each containing unique modiications (I = + 83 Da; S = + 130 Da, (Scheme 4.3)) corresponding to the portion of the cross-link remaining on the peptide side-chain following cleavage of the sulfonium ion (Figure 4.9b). As shown in Scheme 4.3, formation of these characteristic product ions occurs via cleavage within the ionic cross-linker, which results in separation of the two peptide chains. These initial product ions formed from the MS/MS experiment were subjected to fragmentation via MS3 to obtain further structural information required for the identiication of the peptide and the modiication site(s) (Figure 4.9c and d). Although not shown here, O N

O

O S

O

O O

N

O

O H2N-Peptide (1)

H2N-Peptide (2)

O Peptide(1)

O

HN

S

Peptide(2)

NH

MS/MS O

O Peptide(1)

N H M+I

S

NH Peptide(2) M+S

SCHEME 4.3 Structure of the ionic cross-linking reagent S-methyl 5,5′-thiodipentanoylhydroxysuccinimide, an intermolecular cross-linked peptide product formed by reaction with this cross-linker, and the selective gas-phase fragmentation reaction of the intermolecular cross-linked peptides.

108

100

400

600

100

% Relative abundance % Relative abundance

O

NH

p ELYENKPRRPYIL

1000

[M+H]+

1200

1400

1600

1800

2000

1200

1400

1600

1800

2000

[M+2H+S]2+ 902.5 [2M+4H+(I–S)]5+

600

800

1000

y9†3+

100

[b12†+H2O]3+

ρ ELYENKPRRPYIL + NH O

†2+ y11†3+ †3+ a12 –H2O y9 2+

y8†3+

200 (d)

S +

[M+2H+I]3+ 586.1

400 (c)

800

MS

NH

O

[2M+4H+(I–S)]5+ [M+2H]2+

% Relative abundance

(b)

p ELYENKPRRPYIL

[M+3H]3+

% Relative abundance

(a)

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

300

y7

400

100

500

y10†*2+ y10†2+

600

b11†*2+

700

y11†2+

b12†*2+

b6†

800

900

1000

[b12†+H2O]2+ y9‡2+ b9‡2+ a9‡2+ ‡2+ y72+y4 y8

400

600

ρ ELYENKPRRPYIL

b12‡2+ * y7

‡2+ y5 a ‡2+ ‡ b13 12 b11‡2+ ‡2+ b6 y11 y7 a11‡2+

800

1000

NH

b8‡

y8‡* b ‡* 9

1200 m/z

O

S

b11‡*

b9‡

1400

1600

1800

2000

FIGURE 4.9 (a) An electrospray ionization mass scan of cross-linked neurotensin (pELYENKPRRPYIL) following reaction with the ionic cross-linking reagent S-methyl 5,5′-thiodipentanoylhydroxy-succinimide. (b) CID product ion mass spectrum of the [2M + 4H + (I–S)]5 + precursor ion, m/z 712.4, of neurotensin containing an intermolecular peptide cross-link. CID-MS3 product ion mass spectra of the (c) [M + 2H + I]3 + and (d) [M + 2H + S]2 + product ions, from panel B. † indicates product ions containing an I-type modiication and ‡ indicates product ions containing an S-type modiication (see Scheme 4.3).

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dead-end cross-linked peptides, present in either their hydrolyzed or unhydrolyzed forms, are identiied readily based on the observation of a characteristic neutral loss. Further characterization of these dead-end cross-linked peptides can be achieved, therefore, by isolating the initial neutral loss product ion then subjecting it to MS3. Tandem mass spectrometric analysis of intra-molecular cross-linked peptides in the ion trap results in initial cleavage of the cross-linker without changing the m/z of the precursor ion, which results in immediate further fragmentation of the peptide backbone to yield b- and y-type ions. Thus, detailed structural information can be obtained by direct analysis of the MS/MS product ion spectrum. The use of this ionic cross-linking reagent in conjunction with multistage tandem mass spectrometry therefore enables the different types of peptide cross-links to be readily distinguished. Furthermore, the solubility of the ionic cross-linker and its stability under aqueous conditions suggests that it holds great promise for future studies aimed at the structural analysis of large proteins or protein complexes. As an alternative to the gas-phase cleavage of cross-linking reagents via CID, infrared multiphoton dissociation (IRMPD) can be employed also to facilitate the identiication of cross-linked peptides. Recently, Gardner et al. have developed a new IR chromogenic cross-linker (IRCX) that incorporates a phosphate functional group into the cross-linking reagent; the phosphate group has a strong infrared absorption at 10.6 µm [159].* Upon infrared irradiation at 10.6 µm, all peptides that contain this chromogenic cross-linker undergo photodissociation and are distinguished from non-modiied peptides by a decrease in ion abundance. The IRCX-containing peptides identiied in this manner can be interrogated further by IRMPD, CID, or by both methods. This new approach establishes, therefore, another promising chemical cross-linking pathway for the structural analysis of biological assemblies.

4.5

CONCLUDING REMARKS

Chemical derivatization methods provide a useful additional tool for protein structural analysis, particularly when coupled with the multistage tandem mass spectrometric capabilities of modern ion trap mass spectrometers. The objective of this chapter was to provide a brief overview of the chemical derivatization strategies that are employed currently to address the challenges associated with protein identiication, characterization, and quantitative analysis as well as for the characterization of protein–protein interactions.

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105. Ong, S.-E.; Blagoev, B.; Kratchmarova, I.; Bach, Kristensen, D.; Steen, H.; Pandey, A.; Mann, M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 2002, 1, 376–386. 106. Julka, S.; Regnier, F. Quantiication in proteomics through stable isotope coding: a review. J. Proteome Res. 2004, 3, 350–363. 107. Kuhn, K.; Prinz, T.; Schafer, J.; Baumann, C.; Scharfke, M.; Kienle, S.; Schwarz, J.; Steiner, S.; Hamon, C. Protein sequence tags: a novel solution for comparative proteomics. Proteomics 2005, 5, 2364–2368. 108. Simons, B.L.; Wang, G.; Shen, R.-F.; Knepper, M.A. In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics. Rapid Commun. Mass Spectrom. 2006, 20, 2463–2477. 109. Shi, Y.; Yao, X. Oxygen isotopic substitution of peptidyl phosphates for modiicationspeciic mass spectrometry. Anal. Chem. 2007, 79, 8454–8462. 110. Thompson, A.; Schäfer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Tandem mass tags: a novel quantiication strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 2003, 75, 1895–1904. 111. Dayon, L.; Hainard, A.; Licker, V.; Turck, N.; Kuhn, K.; Hochstrasser, D.F.; Burkhard, P.R.; Sanchez, J.-C. Relative quantiication of proteins in human cerebrospinal luids by MS/MS using 6-plex isobaric tags. Anal. Chem. 2008, 80, 2921–2931. 112. Ross, P.L.; Huang, Y.N.; Marchese, J.N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3, 1154–1169. 113. Sachon, E.; Mohammed, S.; Bache, N.; Jenson, O.N. Phosphopeptide quantitation using amine-reactive tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis. Rapid Commun. Mass. Spectrom. 2006, 20, 1127–1134. 114. Choe, L.; D’Ascenzo, M.; Relkin, N.R.; Pappin D.; Ross, P.; Williamson, B.; Guertin, S.; Pribil, P.; Lee, K.H. 8-Plex quantitation of changes in cerebrospinal luid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzeimer’s disease. Proteomics 2007, 7, 3651–3660. 115. Li, S.; Zeng, D. CILAT – A new reagent for quantitative proteomics. Chem. Commun. 2007, 21, 2181–2183. 116. Jones, A.M.E.; Bennett, M.H.; Mansield, J.W.; Grant, M. Analysis of the defence phosphoproteome of Arabidopsis thaliana using differential mass tagging. Proteomics 2006, 6, 4155–4165. 117. Champion, P.A.D.; Stanley, S.A.; Champion, M.M.; Brown, E.J.; Cox, J.S. C-Terminal signal sequence promotes virulence secretion in mycobacterium tuberculosis. Science 2006, 313, 1632–1636. 118. Corvey, C.; Koetter, P.; Beckhaus, T.; Hack, J.; Hofmann, S.; Hampel, M.; Stein, T.; Karas, M.; Entian, K.-D. Carbon source-dependent assembly of the Snf1p kinase complex in Candida albicans. J. Biol. Chem. 2005, 280, 25323–25330. 119. Williamson, B.L.; Marchese, J.; Morrice, N.A. Automated identiication and quantiication of protein phosphorylation sites by LC-MS on a hybrid triple quadrupole linear ion trap mass spectrometer. Mol. Cell. Proteomics 2006, 5, 337–346. 120. Wolf-Yadlin, A.; Hautaniemi, S.; Lauffenburger, D.A.; White, F.M. Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5860–5865.

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121. Cunningham, C.; Glish, G.L. High amplitude short time excitation: a method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 2006, 17, 81–84. 122. Meany, D.L.; Xie, H.; Thompson, L.V.; Arriaga, E.A.; Grifin, T.J. Identiication of carbonylated proteins from enriched rat skeletal muscle mitochondria using afinity chromatography-stable isotope labeling and tandem mass spectrometry. Proteomics 2007, 7, 1150–1163. 123. Grifin, T.J.; Xie, H.; Bandhakavi, S.; Popko, J.; Mohan, A.; Carlis, J.V.; Higgins, L. iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J. Proteome Res. 2007, 6, 4200–4209. 124. Bantscheff, M.; Eberhard, D.; Abraham, Y.; Bastuck, S.; Boesche, M.; Hobson, S.; Mathieson, T.; Perrin, J.; Raida, M.; Rau, C.; Reader, V.; Sweetman, G.; Bauer, A.; Bouwmeester, T.; Hopf, C.; Kruse, U.; Neubauer, G.; Ramsden, N.; Rick, J.; Kuster, B.; Drewes, G. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 2007, 25, 1035–1044. 125. Chang, B.; Ünlü, M.; Clauser, K.; Carr, S.A. iTRAQ-IT: Implementation of iTRAQ quantitation tags on ion trap instruments via MS3. Proc. 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, Texas, 2005. 126. Froelich, J.M.; Kaplinghat, S.; Reid, G.E. Automated neutral loss and data dependent energy resolved “pseudo MS3” for the targeted identiication, characterization and quantitative analysis of methionine-containing peptides. Eur. J. Mass Spectrom. 2008, 14, 219–229. 127. Englander, S.W. Hydrogen exchange and mass spectrometry: a historical perspective. J. Am. Soc. Mass Spectrom. 2006, 17, 1481–1489. 128. Kiselar, J.G.; Maleknia, S.D.; Sullivan, M.; Downard, K.M.; Chance, M.R. Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry. Int. J. Radiat. Biol. 2002, 78, 101–114. 129. Sharp, J.S.; Becker, J.M.; Hettich, R.L. Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry. Anal. Chem. 2004, 76, 672–683. 130. McClintock, C.; Kertesz, V.; Hettich, R.L. Development of an electrochemical oxidation method for probing higher order protein structure with mass spectrometry. Anal. Chem. 2008, 80, 3304–3317. 131. Carruthers, N.J.; Stemmer, P.M. Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation. Biochemistry 2008, 47, 3085–3095. 132. Azim-Zadeh, O.; Hillebrecht, A.; Linne, U.; Marahiel, M.A.; Klebe, G.; Lingelbach, K.; Nyalwidhe, J. Use of biotin derivatives to probe conformational changes in proteins. J. Biol. Chem. 2007, 282, 21609–21617. 133. Ladner, C.L.; Turner, R.J.; Edwards, R.A. Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility. Protein Sci. 2007, 16, 1204–1213. 134. Leitner, A.; Linder, W. Functional probing of arginine residues in proteins using mass spectrometry and an arginine-speciic covalent tagging concept. Anal. Chem. 2005, 77, 4481–4488. 135. Mendoza, V.L.; Vachet, R.W. Protein surface mapping using diethylpyrocarbonate with mass spectrometric detection. Anal. Chem. 2008, 80, 2895–2904. 136. Sinz, A. Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. Mass Spectrom. Rev. 2006, 25, 663–682. 137. Trakselis, M.A.; Alley, S.C.; Ishmael, F.T. Identiication and mapping of protein-protein interactions by a combination of cross-linking, cleavage, and proteomics. Bioconj. Chem. 2005, 16, 741–750.

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138. Young, M.M.; Tang, N.; Hempel, J.C.; Oshiro, C.M.; Taylor, E.W.; Kuntz, I.D.; Gibson, B.W.; Dollinger, D. High throughput protein fold identiication by using experimental constraints derived from intramolecular cross-links and mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802–5806. 139. Alley, S.C.; Ishmael, F.T.; Jones, A.D.; Benkovic, S.J. Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photo-cross-linking and afinity reagent. J. Am. Chem. Soc. 2000, 122, 6126–6127. 140. Trester-Zedlitz, M.; Kamada, K.; Burley, S.K.; Fenyo, D.; Chait, B.T.; Muir, T.W. A modular cross-linking approach for exploring protein iInteractions. J. Am. Chem. Soc. 2003, 125, 2416–2425. 141. Sinz, A.; Kalkhof, S.; Ihling, C. Mapping protein interfaces by a trifunctional crosslinker combined with MALDI-TOF and ESI-FTICR mass spectrometry. J. Am. Soc. Mass Spectrom. 2005, 16, 1921–1931. 142. Ahrends, R.; Kosinski, J.; Kirsch, D.; Manelyte, L.; Giron-Monzon, L.; Hummerich, L.; Schulz, O.; Spengler, B.; Friedhoff, P. Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, afinity puriication, chemical coding and mass spectrometry. Nucl. Acids Res. 2006, 34, 3169–3180. 143. Chowdhury, S.M.; Munske, G.R.; Tang, X.; Bruce, J.E. Collisionally activated dissociation and electron capture dissociation of several mass spectrometry-identiiable chemical cross-linkers. Anal. Chem. 2006, 78, 8183–8193. 144. Reynolds, K.J.; Yao, X.; Fenselau, C. Proteolytic 18O labeling for comparative proteomics: evaluation of endoprotease Glu-C as the catalytic agent. J. Proteome Res. 2002, 1, 27–33. 145. Back, J.W.; Notenboom, V.; de Koning, L.J.; Muijsers, A.O.; Sixma, T.K.; de Koster, C.G.; de Jong, L. Identiication of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling. Anal. Chem. 2002, 74, 4417–4422. 146. Chen, X.; Chen, Y.H.; Anderson, V.E. Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry. Anal. Biochem. 1999, 273, 192–203. 147. Taverner, T.; Hall, N.E.; O’Hair, R.A.J.; Simpson, R.J. Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry. J. Biol. Chem. 2002, 277, 46487–46492. 148. Müller, D.R.; Schindler, P.; Towbin, H.; Wirth, U.; Voshol, H.; Hoving, S.; Steinmetz, M.O. Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 2001, 73, 1927–1934. 149. Pearson, K.M.; Pannell, L.K.; Fales, H.M. Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method. Rapid Commun. Mass Spectrom. 2002, 16, 149–159. 150. Seebacher, J.; Mallick, P.; Zhang, N.; Eddes, J.S.; Aebersold, R.; Gelb, M.H. Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing. J. Proteome Res. 2006, 5, 2270–2282. 151. Petrotchenko, E.V.; Olkhovik, V.K.; Borchers, C.H. Coded cleavable cross-linker for studying protein-protein interaction and protein complexes. Mol. Cell. Proteomics 2005, 4, 1167–1179. 152. Bennett, K.L.; Kussmann, M.; Björk, P.; Godzwon, M.; Mikkelsen, M.; Sørensen, P.; Roepstorff, P. Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping—a novel approach to assess intermolecular protein contacts. Protein Sci. 2000, 9, 1503–1518. 153. Back, J.W.; Sanz, M.A.; de Jong, L.; de Koning, L.J.; Nijtmans, L.G.J.; de Koster, C.G.; Grivell, L.A.; van der Speck, H.; Muijsers, A.O. A structure for the yeast prohibitin complex: structure prediction and evidence from chemical crosslinking and mass spectrometry. Protein Sci. 2002, 11, 2471–2478.

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154. Peterson, J.J.; Young, M.M.; Takemoto, L.J. Probing α-crystallin structure using chemical cross-linkers and mass spectrometry. Molecul. Vision 2004, 10, 857–866. 155. Back, J.W.; Hartog, A.F.; Dekker, H.L.; Muijsers, A.O.; de Koning, L.J.; de Jong, L. A new cross-linker for mass spectrometric analysis of the quaternary structure of protein complexes. J. Am. Soc. Mass Spectrom. 2001, 12, 222–227. 156. Tang, X.; Munske, G.R.; Siems, W.F.; Bruce, J.E. Mass spectrometry identiiable cross-linking strategy for studying protein-protein interactions. Anal. Chem. 2005, 77, 311–318. 157. Soderblom, E.J.; Goshe, M.B. Collision-induced dissociative chemical cross-linking reagents and methodology: applications to protein structural characterization using tandem mass spectrometry analysis. Anal. Chem. 2006, 78, 8059–8068. 158. Lu, Y.; Tanasova, M.; Borhan, B.; Reid, G.E. An ionic reagent for controlling the gas-phase fragmentation reactions of cross-linked peptides. Anal. Chem. 2008, 80, 9279–9287. 159. Gardner, M.W.; Vasicek, L.A.; Shabbir, S.; Anslyn, E.V.; Brodbelt, J.S. Chromogenic cross-linker for the characterization of protein structure by infrared multiphoton dissociation mass spectrometry. Anal. Chem. 2008, 80, 4807–4819.

Transform Ion 5 Fourier Cyclotron Resonance Mass Spectrometry in the Analysis of Peptides and Proteins Helen J. Cooper CONTENTS 5.1

5.2

Fourier Transform Ion Cyclotron Resonance (FT-ICR) ............................... 122 5.1.1 Principles of Fourier Transform Ion Cyclotron Resonance (FT-ICR) ........................................................................................... 123 5.1.1.1 Ion Motion.......................................................................... 123 5.1.1.2 Excitation and Detection .................................................... 125 5.1.2 Instrumentation ................................................................................. 126 5.1.2.1 Magnet ............................................................................... 126 5.1.2.2 Ionization ........................................................................... 127 5.1.2.3 Ion Transfer ........................................................................ 128 5.1.2.4 Ion Cyclotron Resonance (ICR) Cell ................................. 128 5.1.3 Features of Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry ........................................................... 128 5.1.3.1 Mass Accuracy ................................................................... 128 5.1.3.2 Resolving Power................................................................. 129 5.1.3.3 Sensitivity........................................................................... 130 Tandem Mass Spectrometry (MS/MS) in Fourier Transform Ion Cyclotron Resonance (FT-ICR) .................................................................... 130 5.2.1 Precursor Ion Isolation...................................................................... 131 5.2.2 Sustained Off-Resonance Irradiation Collision-Induced Dissociation (SORI-CID) ................................................................. 132 5.2.2.1 Principles of Sustained Off-Resonance Irradiation Collision-Induced Dissociation (SORI-CID) ..................... 132 5.2.2.2 Sustained Off-Resonance Irradiation Collision-Induced Dissociation (SORI-CID) of Peptides and Proteins ............133

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5.2.3

Infrared Multiphoton Dissociation (IRMPD) ................................... 133 5.2.3.1 Principles of Infrared Multiphoton Dissociation (IRMPD) ............................................................................ 133 5.2.3.2 Infrared Multiphoton Dissociation (IRMPD) of Peptides and Proteins ......................................................... 133 5.2.4 Blackbody Infrared Radiative Dissociation (BIRD) ........................ 134 5.2.4.1 Principles of Blackbody Infrared Radiative Dissociation (BIRD) .......................................................... 134 5.2.4.2 Blackbody Infrared Radiative Dissociation (BIRD) of Peptides and Proteins ......................................................... 134 5.2.5 Electron Capture Dissociation (ECD) .............................................. 135 5.2.5.1 Principles of Electron Capture Dissociation (ECD) .......... 135 5.2.5.2 Electron Capture Dissociation (ECD) of Peptides and Proteins .............................................................................. 135 5.2.5.3 Activated Ion Electron Capture Dissociation (AI-ECD) .......................................................................... 137 5.3 Hybrid Fourier Transform Ion Cyclotron Resonance (FT-ICR) Instruments ................................................................................................... 138 5.3.1 Quadrupole-Fourier Transform Ion Cyclotron Resonance (FT-ICR) ........................................................................................... 138 5.3.2 Linear Ion Trap-Fourier Transform Ion Cyclotron Resonance (FT-ICR) ........................................................................................... 139 5.4 Applications of Fourier Transform Ion Cyclotron Resonance (FT-ICR) in Proteomics ................................................................................................ 139 5.4.1 ‘Bottom-Up’ Approaches .................................................................. 139 5.4.1.1 Peptide Mass Fingerprinting .............................................. 139 5.4.1.2 Peptide Sequencing ............................................................ 140 5.4.2 ‘Top-Down’ Approaches................................................................... 143 5.5 Summary ...................................................................................................... 144 References .............................................................................................................. 145

5.1

FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR)

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [1,2] has developed due to the fact that a charged particle in a uniform magnetic ield will undergo cyclotron motion, that is, will describe a circular path, perpendicular to the direction of the magnetic ield, and the frequency of that motion is inversely proportional to its mass-to-charge ratio. The technique of FT-ICR mass spectrometry offers the highest resolution and mass accuracy of all mass analyzers, making it ideal for the characterization of peptides and proteins. This chapter provides an overview of FT-ICR mass spectrometry and its applications in structural characterization of peptides and proteins. The principles of FT-ICR (ion motion, excitation/detection, and instrumental considerations) are discussed together with the features of FT-ICR that make it so suitable for peptide/protein analysis. Tandem mass spectrometry (MS/MS) techniques (sustained off-resonance irradiation collision-induced dissociation (SORI-CID), infrared

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multiphoton dissociation (IRMPD), black body infrared radiative dissociation (BIRD), and electron capture dissociation (ECD) for the sequencing of peptides/proteins are described; see Section 5.2 for an explanation of these acronyms. The new generation of hybrid FT-ICR instruments are reviewed. Finally, the chapter includes a discussion of the applications of FT-ICR in ‘bottom-up’ and “top-down” proteomics.

5.1.1

PRINCIPLES OF FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR)

5.1.1.1 Ion Motion As described above, an ion moving in the presence of a uniform magnetic ield, B, experiences the Lorentz force: F = ma = qvB

(5.1),

where m is the mass of the ion, a is the acceleration experienced by the ion, q is the charge of the ion, and v its velocity. The Lorentz force is directed perpendicular to the direction of the ion’s velocity and to the magnetic ield. Consequently, in the absence of any collisions, the ion’s trajectory is curved into a circle of radius, r, that is, the ion acquires radial velocity in the xy plane (see Figure 5.1). The angular acceleration of the ion is given by: a=

2 v xy r

(5.2),

z x y B Lorentz force

B

v

ω

FIGURE 5.1 Ion cyclotron motion. The ion describes a circular path perpendicular to the direction of the magnetic ield.

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where vxy is the velocity of the ion in the xy plane. The angular frequency of the ion, ω, is: ω=

v xy r

(5.3).

Rearranging Equations 5.1 through 5.3, we get the cyclotron equation ω=

qB m

(5.4).

The cyclotron equation shows that the frequency at which an ion undergoes cyclotron motion is inversely proportional to its mass-to-charge ratio. Thus, when the cyclotron frequency is measured, m/z may be calculated. Although ions are trapped in the plane perpendicular to the magnetic ield, they are not trapped in the direction parallel to the magnetic ield. It is necessary, therefore, to apply a trapping potential to two electrodes at either end of the ion cyclotron resonance (ICR) cell. The electric ield that is produced is three-dimensional and non-linear because the trapping electrodes are inite in size. Two additional types of ion motion are observed as a consequence of the electrostatic ield (see Figure 5.2). The ion will move back and forth parallel to the direction of the magnetic ield; this motion is known as trapping oscillation. The frequency of trapping oscillation depends on the size and shape of the ICR cell. The trapping potential also has a radial component that produces an outward electrical force on the ion (cf the inwarddirected Lorentz force). The radial force on the ion is F=

qVtrap α r d2

(5.5),

where Vtrap is the trapping potential, α is a constant determined by the ICR cell geometry, and d is the distance between the trapping electrodes. As a consequence, the ion will undergo magnetron motion. The center of the cyclotron motion will describe a circular path perpendicular to the direction of the magnetic ield. Magnetron motion and trapping oscillation frequencies are much smaller than the cyclotron frequency and are not detected typically in FT-ICR. z x y Cyclotron motion

Magnetron motion

Trapping oscillation

FIGURE 5.2 The three modes of motion undertaken by an ion trapped in an ICR cell: cyclotron motion, magnetron motion, and trapping oscillation.

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As described, the radial electric force opposes the Lorentz force. Thus the overall force acting on the ion is now: Force = mω2r = qωrB −

qVtrap α r d2

(5.6).

Rearranging Equation 5.6 gives a quadratic equation in ω that can be solved to give the reduced cyclotron frequency ω=

ωc + 2

( ω2 ) − ω2 2

c

2 z

(5.7),

where ωc is the cyclotron frequency and ωz is the trapping oscillation frequency. As a result, the frequency-to-m/z calibration equation [3] is non-linear. It is necessary when calibrating, therefore, to use at least two m/z-values. Ideally, multiple m/z-values should be used as cyclotron motion is also affected by Coulombic repulsion between ions. 5.1.1.2 Excitation and Detection The goal of FT-ICR mass spectrometry is to measure the cyclotron frequency, by detecting the image current as a packet of ions passes by a detector plate, and thus calculate m/z. This goal cannot be achieved, however, simply by trapping ions in an ICR cell. First, ions of the same m/z-value are not necessarily in phase with each other. On injection to the ICR cell, an ion can start its cyclotron motion at any point on the cyclotron path. The net image current induced by two ions ‘opposite’ each other, that is, 180° out of phase, will be zero. Second, the cyclotron motion needs to be at a radius suficiently large to permit detection. For example, at room temperature, a singly-charged ion of mass 1000 Da has a cyclotron radius of 0.08 mm in a 9.4 T magnetic ield [2]. The diameter of the ICR cell is of the order of 10 cm. In order to detect ions trapped in an ICR cell, it is necessary irst to induce coherent motion, that is, ions of the same m/z need to be traveling together in a single ion packet at a radius close to that of the ICR cell. Coherent motion is achieved by exciting the ions

Excite

Detect

Detect

Excite

FIGURE 5.3 Excitation of an ion trapped in an ICR cell.

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100

200

300 400 500 Time (ms)

600

700

800

Fourier transform

150 200 Frequency (kHz)

100

Frequency to m/z calibration

12+ 600

800

250

7+ 1000

1200 1400 m/z

1600

1800

2000

FIGURE 5.4 FT-ICR analysis of electrosprayed ubiquitin. Detection of the image current results in the time-domain signal (top). Fourier transformation converts the time-domain signal to a frequency spectrum (middle) that is calibrated to give the mass spectrum (bottom).

via application of an oscillating electric ield at, or near, the cyclotron frequency of the ions. A radiofrequency potential containing frequencies that span the desired m/z-range is applied to two of the ICR electrodes, see Figure 5.3. Ions orbiting at these frequencies absorb energy; their kinetic energy is increased and, hence, their cyclotron radius. The cyclotron radius following excitation is independent of m/z; all ions of a given m/z-range are excited to the same radius without any mass discrimination effects. Post-excitation, a second pair of electrode is used to detect the image current as the clouds of ions cycle around the cell. The resulting time-domain signal comprises the superimposed signals from each of the ion packets. A Fourier transform is applied to convert the time-domain signal into a frequency spectrum, that is, to ‘pull out’ each frequency in the complex signal [4]. A frequency-to-m/z calibration, based on Equations 5.4 and 5.7, is performed resulting in a mass spectrum as depicted in Figure 5.4. Figure 5.4 shows the FT-ICR analysis of electrosprayed ubiquitin, a small protein of ca 8.5 kDa. The mass spectrum reveals the presence of charge states + 7 through + 12, that is, peaks corresponding to [M + 7H]7 + through [M + 12H]12 + .

5.1.2

INSTRUMENTATION

5.1.2.1 Magnet A permanent magnet, the homogeneity of which is crucial for satisfactory performance, is central to the FT-ICR mass spectrometer. Commercial instruments featuring

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superconducting magnets of ield strength 3–15 T are available. Several parameters improve with magnetic ield strength; these include mass resolving power, signalto-noise ratio, dynamic range, and mass accuracy [5]. There is also a concomitant increase in price! To address the problem of stray magnetic ield, the magnet may be shielded either passively, for example, enclosed within steel, or actively. Active shielding involves a second set of coils outside the main magnet having a ield in the opposing direction, thereby canceling the external magnetic ield. 5.1.2.2 Ionization Most FT-ICR applications involving the analysis of peptides and proteins utilize electrospray ionization (ESI) [6]. It is possible also to couple matrix-assisted laser desorption/ionization (MALDI) with FT-ICR mass spectrometers, and these instruments are available commercially, however, they will not be discussed in detail here. 5.1.2.2.1 Electrospray Ionization (ESI) The techniques of ESI and MALDI have revolutionized biological mass spectrometry by enabling the generation of intact either protonated or deprotonated molecules from peptides, proteins, oligonucleotides, and sugars. So great has been their impact that the scientists behind their development, John Fenn (ESI) and Koichi Tanaka (MALDI), were awarded the Nobel Prize for Chemistry in 2002. Prior to the introduction of these techniques, it was not possible to investigate biological molecules greater than ca 2–3 kDa. In ESI [7], a solution containing the analyte lows through a capillary at the mouth of the mass spectrometer. The solution comprises typically an aqueous/organic mix such as 1:1 water:acetonitrile. In positive electrospray, that is, the generation of cations, the solution is acidiied with formic or acetic acid (≤ 2%). Generation of anions (negative electrospray) involves typically addition of a base to the solution, for example, ammonium hydroxide. In experiments where it is necessary to preserve non-covalent interactions, for example, when analyzing a peptide:protein complex, the analyte may be sprayed from a solution containing a volatile buffer such as ammonium acetate at a concentration of 5–10 mM. It is very important to note, when considering the ESI of peptides and proteins, that either a high salt concentration or a solution containing detergent will affect adversely the ESI of the analyte. A potential is applied between the tip of the capillary and the entrance to the mass spectrometer, the result of which is dispersal of the solution into ine, charged droplets. Note that ESI is an atmospheric pressure interface. The charged droplets are swept toward the entrance to the mass spectrometer by the pressure gradient. As the droplets proceed, the solvent desorbs and the droplets decrease in size until the repulsion between the charges on the surface of the droplet is such that a Coulombic explosion occurs producing many smaller droplets. The process repeats itself until eventually gas-phase analyte ions remain. It is possible to enhance this process by applying low lows of nitrogen gas to encourage desorption of the solvent. The number of charges on the analyte ions depends on the nature of the analyte and of the solution. Positive electrospray of peptides and proteins tends to result in multiply-charged ions (see Figure 5.4), in which the charges are associated with basic amino acid residues (lysine, arginine, and histidine) and the N-terminus.

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Electrospray was irst coupled with FT-ICR mass spectrometry in 1989 [8], and the two are very well-suited, particularly in the analysis of large biomolecules. ESI of a protein results in multiply-charged ions, many of which lie within the m/z-range of the FT-ICR mass spectrometer (ca 50–5000 Th). The high resolving power associated with FT-ICR allows the complex isotopic distributions that exist within each charge state to be scrutinized. 5.1.2.3 Ion Transfer As mentioned above, ESI is an atmospheric pressure process whereas the pressure in the ICR cell is of the order of 10−9 Torr. It is necessary, therefore, to transport the ions generated through a succession of differentially-pumped regions of lower pressure. In addition, the ESI source is situated usually outside of the magnetic ield and the ions must be transported along the magnetic ield lines. The ions produced by electrospray have both axial (parallel to the magnetic ield) and radial (perpendicular to the magnetic ield) velocity components. As the ions are transported, they experience a large magnetic ield gradient. As the magnetic ield strength increases, the radial velocity increases. Since kinetic energy must be conserved, the axial velocity decreases. Depending on the initial ratio of the axial to radial velocities, this phenomenon can lead to the magnetic mirror effect, in which the ions stop their forward motion and reverse without reaching the ICR cell. To avoid the magnetic mirror effect, either the ions are accelerated to a very high axial velocity by use of electrostatic lenses [9] or focusing devices such as radiofrequency (RF)-quadrupoles [10], hexapoles [11], or octopoles [12] are used to transport the ions. ESI is a continuous process. While the potential is applied between the capillary tip and the entrance to the mass spectrometer, ions are generated in a constant stream. However, the nature of the FT-ICR experiment requires discrete packets of ions that are irst trapped in the ICR cell then excited and detected. To maximize the duty cycle, ions are accumulated externally before being transferred to the ICR cell [13]. Accumulation of ions can be achieved by use of storage hexapoles, octapoles, or ion traps. 5.1.2.4 Ion Cyclotron Resonance (ICR) Cell The principal component of an FT-ICR mass spectrometer is the ICR cell, located in the magnetic ield in which the ions are trapped. The axis of the cell is aligned with the magnetic ield. A number of different cell geometries are in use including cubic, cylindrical with end-caps, open cylindrical, and ‘matrix-shimmed’ [14]. The cylindrical design is particularly widespread. The effects of the cell geometry on ion motion are discussed above.

5.1.3

FEATURES OF FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR) MASS SPECTROMETRY

5.1.3.1 Mass Accuracy FT-ICR mass spectrometry offers unprecedented mass accuracy. This feature is a consequence of the inherent accuracy of frequency measurement. Sub-ppm mass accuracies are obtained routinely. It is possible, up to ca m/z 400, to assign elemental composition (CcHhNnOoPpSs) based on mass alone. This approach has been applied to the ields of

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petroleomics [15] and metabolomics [16]. Smith and co-workers have developed methods in which the mass accuracy associated with FT-ICR is applied to protein identiication in proteomics [17,18]. The accurate mass tag (AMT), approach was applied to the analysis of Deinococcus radiodurans identifying over 60% of the predicted proteome [19]. The mass accuracy allows direct determination of small modiications in large intact protein ions, for example, the presence of a disulide bond (−2 Da). In peptide analysis, it is possible to distinguish between amino acids glutamine and lysine (36 mDa). 5.1.3.2 Resolving Power FT-ICR mass spectrometry offers ultrahigh resolution. This feature is a result of the large numbers of cyclotron orbits during detection and the fact that cyclotron frequency is independent of ion velocity. Performance is not limited by the initial position, direction or speed of the ions, unlike mass spectrometers such as time-oflight or sector instruments. FT-ICR resolution can be deined as the full width of a spectral peak at half of the maximum peak height, that is, ∆m50% for mass spectra, or ∆ω50% for frequency domain spectra. The FT-ICR resolving power is deined as m/∆m50% or ω/∆ω50%. The irst derivative of Equation 5.4 gives m qB =− ∆ m50% m∆ω 50%

(5.8).

Provided that the peak width of the frequency domain spectral peak is independent of magnetic ield, that is, post-excitation ion kinetic energy is constant, the FT-ICR resolving power increases linearly with increasing magnetic ield. For a constant magnetic ield, FT-ICR resolving power varies inversely with m/z. At the low pressure limit, that is, when the time-domain signal persists undamped throughout the acquisition period, the mass spectral peak width is independent of m/z. As m/z increases, the peak width remains constant, however, because ICR frequency varies inversely with m/z, the peaks are more closely spaced. The resolving power associated with FT-ICR has a number of advantages in the analysis of biomolecules. Unit mass resolution has been demonstrated for proteins up to 112 kDa [20], that is, it is possible to distinguish between isotopic peaks containing 13C, 15N, 18O, 34S, etc. The ability to distinguish between isotopic peaks allows direct determination of charge state. The nominal mass difference between isotopic ions is 1 Da, for example, one 12C versus one 13C. The difference in m/z-spacing between isotopic ion peaks corresponds to 1/z. When an ion is in the + 12 charge state, the spacing between the isotopic peaks in the mass spectrum will be 1/12 Th. When it is in the + 6 charge state, the spacing will be 1/6 Th. As protein ions increase in molecular weight, it becomes increasingly statistically likely that the monoisotopic peak will not be observed. Averagine has the molecular formula C4.9384H7.7583N1.3577O1.4773S0.0417, molecular mass 111.1254 Da, and is based on the statistical occurrence of common amino acids. By itting the observed isotopic distribution against a model distribution based on averagine amino acid residues, it is possible to calculate the monoisotopic mass [21]. In addition to resolving isotopomers, it is possible to resolve isotopic ine structure, for example, to distinguish between two isotopic ions where one ion contains

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two 13C atoms and one ion contains one 34S atom, that is, (Cc-213C2HhOoNnSs) versus (CcHhOoNnSs-134S) by FT-ICR mass spectrometry. Marshall and co-workers demonstrated resolution of isotopic ine structure resolution in a protein of 15.8 kDa [22]. In terms of peptide analysis, the resolving power of FT-ICR allows resolution of phosphorylated and sulfated peptides, PH versus S, a mass difference of 9.5 mDa [23]. In a truly elegant demonstration of resolving power, two peptides of nominal mass 904 Da, but differing in elemental composition by N4O versus S2H8, were baseline-resolved [24]. The actual difference in mass between these peptides was 0.45 mDa – less than the mass of an electron (0.54 mDa)! 5.1.3.3 Sensitivity The sensitivity of FT-ICR mass spectrometry is limited by image current detection. Approximately, 100 charges are required to generate a measurable frequency for a given value of m/z. ESI is advantageous as multiply-charged ions are generated, however, ESI is concentration-sensitive and the analysis of dilute samples can be problematic. Further problems arise because the generated ions need to be transported some distance through a pressure gradient starting at atmospheric pressure and ending at around 10−9 Torr. It is often the case that FT-ICR is interfaced with an on-line separation technique such as nano-liquid chromatography (LC) [25] when analyzing samples of low concentration and high complexity. This coniguration concentrates the sample before it enters the mass spectrometer and has the additional advantage of on-line desalting. It should be noted that the presence of salt is common in biological samples and interferes with the electrospray process. Reproducible detection of 100 amol (1 µL of a 100 amol µL −1 solution loaded on column) and 300 amol (1 µL of a 300 amol µL −1 solution) of a single peptide in water and in artiicial cerebrospinal luid, respectively, have been demonstrated [11]. Ten amol (0.5 µL of a 20 amol µL −1 solution) of cytochrome C peptides in a six-protein mix have been detected by this method [26].

5.2

TANDEM MASS SPECTROMETRY (MS/MS) IN FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR)

In order to gain sequence information about a peptide or protein, it is necessary to fragment the ion in a sequence-speciic manner, that is, to cleave between each amino acid residue with a single cleavage per intact peptide or protein ion. A mixture of fragments, corresponding to all possible cleavages, will result and the sequence can be deduced from the mass differences between the fragments. The nomenclature for describing the fragments of peptide/protein ions was devised by Roepstorff et al. [27] and is illustrated in Figure 5.5. Fragmentation of peptide and protein ions in FT-ICR mass spectrometry may be induced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) [28], infrared multiphoton dissociation (IRMPD) [29,30], blackbody infrared radiative dissociation (BIRD) [31,32], surface-induced dissociation (SID) [33,34], and electron capture dissociation (ECD) [35,36]. These techniques are ‘true’ MS/MS techniques in which the precursor ion is isolated prior to fragmentation. Additional techniques in which ions are not isolated but fragmented before they

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a b c

R1 H2 N O

R3

O

H N R2

N H

O

H N O

OH R4

x y z

FIGURE 5.5 Nomenclature for describing the backbone product ions of peptide/protein ions. (Reproduced from Roepstorff, P.; Fohlman, J., Biol. Mass Spectrom. 1984, 11, 601.)

reach the ICR cell include multipole storage-assisted dissociation (MSAD) [37,38] and nozzle-skimmer dissociation [39].

5.2.1

PRECURSOR ION ISOLATION

Isolation of precursor ions for FT-ICR MS/MS can take place either prior to the ions entering the ICR cell or after trapping of the ions in the cell, that is, FT-ICR MS/ MS can be spatial or temporal. Temporal MS/MS involves resonant excitation of the unwanted ions in the ICR cell; here, all but the required precursor ions are removed from the cell. This process is similar to that used to excite the ions to increased cyclotron orbits prior to image current detection. For the removal of ions, however, the ions are excited to a cyclotron radius greater than the dimensions of the ICR cell. Once the ions collide with the cell walls, they are ‘ejected’ from the trap. As described above, resonant excitation of ions prior to detection involves application to two of the ICR electrodes of a radiofrequency potential containing frequencies which span the desired m/z-range. In precursor isolation, the applied potential contains all frequencies except those corresponding to the ions to be isolated. An elegant feature of this approach is that it is possible to perform ‘double notch’ isolation where two (or more) ions of quite separate m/z can remain in the ICR cell. One method for temporal precursor isolation is ‘stored waveform inverse Fourier transform’ (SWIFT) [40]. In this method, the desired frequency domain proile (all frequencies except that of the ion of interest) is inversely Fourier transformed to a time domain waveform. This waveform is then applied to the ‘excite’ electrodes in the ICR cell and, thus, the precursor ions are isolated in the cell. An alternative technique for in-cell isolation is correlated sweep excitation (COSE) [41], also known as correlated harmonic excitation ields (CHEF) [42]. This method involves application of radiofrequency pulses to the ‘excite’ electrodes. The technique correlates the duration and frequency of the RF pulses with those appropriate to the ions to be isolated. Both SWIFT and COSE are capable of isolating single isotopomers in peptide and protein ions [43–45]. As described above, it is possible also to isolate precursor ions prior to entry to the ICR cell. Hybrid FT-ICR instruments, which are interfaced with front-end

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resolving quadrupoles, were developed in the groups of Marshall [46,47] and Smith [48,49] and are now available commercially. These instruments allow mass-selective external accumulation of ions. Similarly, FT-ICR instruments interfaced with linear ion traps are available commercially also. In these instruments, ion isolation occurs solely at the front-end—there is no in-cell selection. Hybrid FT-ICR instruments are discussed in more detail below.

5.2.2

SUSTAINED OFF-RESONANCE IRRADIATION COLLISION-INDUCED DISSOCIATION (SORI-CID)

5.2.2.1 Principles of Sustained Off-Resonance Irradiation Collision-Induced Dissociation (SORI-CID) Collision-induced dissociation (CID) [50] is the mainstay of MS/MS. The technique involves collisions between translationally-excited ions with inert gas atoms or molecules. Translational energy is converted to internal energy as a result of the inelastic collision. ‘Slow heating’ of the precursor ions to a higher Boltzmann temperature results in formation of product ions by the lowest energy pathway(s). In peptides and proteins, cleavage of the peptide bond occurs resulting in b and y fragments (see Figure 5.5). The irst obstacle for FT-ICR-based CID, that the ions must be excited translationally, may be surmounted by resonant excitation of the ions (as in pre-detection excitation, or radial ejection, described above). However, the extent of excitation is limited by the magnetic ield and the size of the trap. Excess excitation would result in ejection of the ions from the ICR cell. A second problem is that the products are formed off-axis because the precursor ions are increasing their cyclotron radius; this effect results in reduced resolving power and prevents any further fragmentation (MSn). SORI-CID, introduced by Gauthier and co-workers [28], is not beset by these problems. As the name suggests, ions are excited slightly off-resonance (500–2000 Hz). Such excitation results in acceleration and deceleration of the ions with a period equal to the difference between the excitation frequency and the ion cyclotron frequency. The periodic decrease in cyclotron radius means that ions are not ejected from the ICR cell. Prior to off-resonance excitation of the precursor ions, inert gas is leaked into the ICR cell. As the ions are excited, collisions with the gas result in conversion of translational energy to internal energy. Again, as a result of the periodic decrease in cyclotron radius, the product ions are formed close to the center of the cell, eliminating resolution issues. It is possible that the product ions have a cyclotron frequency equal to that of the applied excitation waveform. If this were the case, those product ions would be ejected from the ICR cell (resonant ejection). To avoid this occurrence, off-resonance excitation is performed in both directions, for example, ±500 Hz. A shortcoming of SORI-CID is that gas must be leaked into the ICR cell. However, high resolution FT-ICR measurements require cell low pressure. It is necessary, therefore, to introduce a delay in the experiment following the SORI-CID event. This delay allows the cell pressure to return to normal (ca 10−9 Torr). The delay is of the order of tens of seconds, making SORI-CID incompatible with on-line separation techniques.

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5.2.2.2

Sustained Off-Resonance Irradiation Collision-Induced Dissociation (SORI-CID) of Peptides and Proteins SORI-CID is a ‘slow-heating’ fragmentation technique in which product ions are formed by the lowest energy pathways. For peptides and proteins, cleavage of the peptide bond occurs to give b and y ions. In addition, losses of small neutral molecules, such as water or ammonia, are observed frequently. Studies of SORI-CID of peptides and proteins have shown that the technique has high eficiency, selectivity, and resolving power [51]. The technique has been applied to peptides [43,45,52–54] and proteins including cytochome c (ca 12 kDa) [55], myoglobin (ca 17 kDa) [45,56], carbonic anhydrase (ca 29 kDa) [57], and monomeric M-CSF (ca 25 kDa) [58]. Multiplestage SORI-CID (up to MS4) has been reported [59]. In addition to providing primary sequence information, SORI-CID can be used to dissociate non-covalent protein complexes [60] and to remove salt from protein ions in the ICR cell [58].

5.2.3

INFRARED MULTIPHOTON DISSOCIATION (IRMPD)

5.2.3.1 Principles of Infrared Multiphoton Dissociation (IRMPD) In IRMPD [29,30], ions are activated by irradiation with photons in the ICR cell. The photons are provided typically by a 10.6 µm CO2 laser. Precursor ions are heated slowly to their dissociation threshold and, as with SORI-CID, they fragment via the lowest energy pathways. The optimum irradiation time for peptides and proteins is 100–200 ms [29]. IRMPD offers a number of advantages over SORI-CID. The principal advantage is that the introduction of gas to the ICR cell is obviated. There is no requirement for a pump-down delay, hence the speed of analysis is greater and the method is compatible with on-line separation techniques [61]. Unlike SORI-CID, blind-spots in the MS/MS spectrum do not occur because there is no resonant excitation of the product ions. All product ions are formed on-axis so there is no loss of resolution and it is possible to undertake further stages of MS/MS. A consequence of the on-axis position is the potential for secondary fragmentation, that is, the product ions can be photon activated also. Fragmentation of product ions can complicate spectral interpretation. 5.2.3.2

Infrared Multiphoton Dissociation (IRMPD) of Peptides and Proteins IRMPD of peptide and protein ions results in cleavage of the peptide bond to give b and y product ions (see Figure 5.5). Greater product ion fragmentation is observed with IRMPD than with SORI-CID. This feature is a consequence of the on-axis nature of the IRMPD technique. Typically, peptide-bond cleavage is more extensive in IRMPD than in SORI-CID. The fact that IRMPD and SORI-CID produce similar product ions, despite being dissimilar activation methods, is indicative of rapid energy partitioning within the precursor ion. An example of an IRMPD mass spectrum of a peptide is given in Figure 5.6; b and y product ions dominate the mass spectrum, with loss of water and ammonia observed also. IRMPD has been applied to peptides and proteins including ubiquitin (ca 8.5 kDa), cytochrome C (ca 12 kDa), carbonic anhydrase (ca 29 kDa), glucokinase (ca 34 kDa),

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×3

ISQAVHAAHAEINEAGR

[M+2H]2+

y3 y7–H2O y6 y4–H2O

b8 y8 b10

b4 y5

y1 y2

200

400

600

b7

y7

800

y9 y 10

y12

y11

b14–NH3

b11 b9

m/z

b14

b12 1000

1200

1400

1600

FIGURE 5.6 IRMPD FT-ICR mass spectrum obtained from [M + 2H]2 + ions of the ovalbumin tryptic peptide ISQAVHAAHAEINEAGR. Precursor ions were irradiated for 80 ms at 60% laser power.

protein A (ca 45 kDa), and serum albumin (ca 67 kDa) [29,62,63]. IRMPD has been applied also to the analysis of ubiquitinated and sumoylated proteins [64,65], and glycoproteins [66,67].

5.2.4

BLACKBODY INFRARED RADIATIVE DISSOCIATION (BIRD)

5.2.4.1 Principles of Blackbody Infrared Radiative Dissociation (BIRD) In BIRD [32], ions are activated by absorption of infrared photons emitted by nearby materials. The vacuum chamber surrounding the ICR cell, when heated normally, emits infrared radiation. This thermal infrared radiation is absorbed by the precursor ions and they are heated close to the temperature of the chamber. As a result, the precursor ions fragment via the lowest energy pathways. Temperatures of up to 500 K are accessible by this method. The rate of dissociation of peptides and proteins at these temperatures is slow and it typically takes 10–1000 s to acquire a BIRD mass spectrum. In addition, long times are needed for the temperature of the ICR cell to equilibrate with that of the vacuum chamber. Unlike SORI-CID, the BIRD technique is neither troubled by the presence of blind-spots in the resulting mass spectra (there is no resonant excitation of product ions), nor are the product ions formed off-axis. 5.2.4.2 Blackbody Infrared Radiative Dissociation (BIRD) of Peptides and Proteins The long timescales associated with BIRD mean that it is not the technique of choice when sequencing peptides and proteins. Moreover, BIRD has been shown to be

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ineficient at protein sequencing when compared with other methods such as CID or ECD [68]. Very low energy fragmentation channels are observed as a result of the slow activation over relatively long timescales. The lowest energy fragmentation channel is often that of extensive water loss. Loss of up to eight water molecules from ubiquitin precursor ions has been demonstrated [32]. Although BIRD is not ideal for peptide/protein sequencing, the fact that the temperature of the ions’ environment can be measured accurately means it is eminently suitable for determining rate constants, activation energies, and dissociation energies.

5.2.5

ELECTRON CAPTURE DISSOCIATION (ECD)

5.2.5.1 Principles of Electron Capture Dissociation (ECD) ECD [35] involves irradiation of precursor ions trapped in the ICR cell with low energy ( 20 kDa), however, few backbone fragments are observed [75]. This effect arises because whereas ECD cleaves the peptide backbone, it does not disrupt non-covalent bonds. The folded protein ion, therefore, remains intact. Activated ion techniques are used to address this problem and are described in more detail below. A second advantage of ECD for peptide/protein analysis is that backbone fragments tend to retain post-translational modiications (PTMs) [76–80], such as phosphorylation and glycosylation. It is possible, therefore, to identify sites of modiication directly. ‘Slow-heating’ techniques tend to result in loss of labile modiications, often [M+2H]2+

FESNFNTQATNR

z11 z2

z5 z4

200

400

600

z6

z7 800 m/z

z8

z9 z10

1000

1200

c11 [M+2H]+ 1400

FIGURE 5.7 ECD FT-ICR mass spectrum of [M + 2H]2 + ions of the lysozyme [52–63] tryptic peptide FESNFNTQATNR.

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[M+2H] + [M+2H]2+ FQ p SEEQQQTEDELQDK

z12

z9 z8 z5

z4

z6

c13 z11

z7

500

c11 c12

z10

1000

m/z

1500

c14

c15

z13

2000

FIGURE 5.8 ECD FT-ICR mass spectrum of [M + 2H]2 + ions of the β-casein phosphopeptide FQpSEEQQQTEDELQDK; p denotes phosphorylation.

at the expense of sequence fragments. It is possible to conirm the presence of the modiication but not necessarily the site. An example of an ECD mass spectrum of a phosphopeptide is shown in Figure 5.8. Although there are two possible sites of phosphorylation in this peptide (Ser3 and Thr9), it is possible to localize the modiication to the serine residue based on the ECD fragmentation pattern. ECD results in a number of minor fragmentation channels in addition to the c/z• cleavage described above. Dissociation to a • and y product ions may also be observed [81]. ECD is unique in that it cleaves disulide bonds [81]. Disulide bonds are not cleaved following CID, and serve as a hindrance to interpretation of CID mass spectra as overlapping series of b and y fragments are observed. ECD results also in the direct loss of amino acid side-chains from the precursor rather than as the result of secondary fragmentation [82]. Peaks corresponding to amino acid side chain loss are similar in abundance to those of c and z• product ions, and can be used diagnostically to conirm the presence of particular amino acid residues. Hot ECD (HECD) [83] is a related technique in which higher energy electrons are utilized. HECD mass spectra are characterized by the presence of secondary product ions arising from the loss of amino acid side chains from z• ions. These secondary ions are useful in distinguishing the isomers leucine and isoleucine. Loss of •CH(CH3)2 (43 Da) is observed from z• ions containing an N-terminal leucine and loss of •CH2CH3 (29 Da) is observed from z• ions containing N-terminal isoleucine. 5.2.5.3 Activated Ion Electron Capture Dissociation (AI-ECD) As described above, ECD of large polypeptides and proteins (> 20 kDa) is characterized by low fragmentation eficiency. Abundant peaks corresponding to chargereduced species but few backbone product ions are observed. Electron capture cleaves

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the peptide backbone but fails to disrupt non-covalent bonds. Similar behavior is seen for smaller tightly-folded species. Activated ion ECD [75,84,85], in which ions are heated prior to, during, or after ECD circumvents this problem. As a result of heating, the ions are unfolded enabling the sequence fragments to dissociate. Activation of ions can be achieved by infrared irradiation, blackbody irradiation, or collisional activation using a nitrogen gas pulse. An alternative approach is plasma ECD [86], in which electrons (0.1−15 eV) are collided with pulsed nitrogen gas prior to the trapping of ions in the ICR cell. The induced plasma conditions result in signiicant increase in ECD eficiency. A single plasma ECD mass spectrum of carbonic anhydrase (ca 29 kDa) showed peaks corresponding to cleavage of 183/253 N–Cα bonds cf 116/258 by activated ion ECD.

5.3

HYBRID FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR) INSTRUMENTS

Since 2000, the ield has moved increasingly toward hybrid FT-ICR instruments in which the FT-ICR is interfaced with a front-end mass analyzer. The groups of Marshall [46,47] and Smith [48,49] introduced the quadrupole-FT-ICR. That coniguration is available commercially. The hybrid linear ion trap FT-ICR [87] was introduced commercially in 2003. Hybrid instruments offer greater versatility in terms of mass-selective external accumulation with the associated increase in sensitivity and dynamic range.

5.3.1

QUADRUPOLE-FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR)

The beneits of the accumulation of ions external to the ICR cell are described above. As ion detection and accumulation are separated physically, one packet of ions is being detected whilst the next packet is being accumulated. The duty cycle, that is, the fraction of time that ions are accumulated for detection, can therefore approach 100%. In addition, external accumulation of ions results in enhanced signal-to-noise ratio and mass resolving power [13]. A factor which limits the maximum achievable duty cycle is the time taken to purge the external trap of ions. Smith and co-workers [48] introduced a 10-cm long segmented accumulation quadrupole that could be purged completely of ions in 400 µs. Marshall and co-workers introduced an alternative approach, in which a direct current (DC) voltage is applied to angled wires positioned between adjacent rods of the accumulation octopole [88]. Further beneits can be realized by the implementation of mass-selective external accumulation. The dynamic range and sensitivity of the instrument are improved. Mass-selective external accumulation can be achieved by interfacing a quadrupole mass ilter with the FT-ICR mass spectrometer. The quadrupole can be operated either in RF/DC mass iltering mode, in which one m/z region traverses the quadrupole, or in RF-only resonant dipolar excitation mode. The latter allows selective removal of multiple m/z peaks. For example, Smith and co-workers showed that this mode could be applied to remove the [M + 16H]16 + and [M + 14H]14 + ions of myoglobin from the charge-state envelope + 13 through + 18 [49].

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5.3.2

139

LINEAR ION TRAP-FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR)

An alternative approach was introduced by Hunt and co-workers [87]. Those researchers coupled a linear quadrupole ion trap, consisting of four rods of hyberbolic crosssection, with an FT-ICR mass spectrometer. The linear ion trap allows accumulation of larger populations of ions than does a standard three-dimensional (3D) ion trap. The hybrid linear ion trap-FT-ICR instrument enables simultaneous detection in both mass analyzers. This aspect is particularly advantageous for ‘data-dependent’ MS/ MS methods used in proteomics, and is discussed further below. The commercial version of this instrument features automated gain control that accumulates a ixed number of charges before delivery to the ICR cell. Because the ‘ideal’ ion density is attained in the cell, space-charge effects resulting in loss of mass resolution and mass accuracy, are eliminated.

5.4

APPLICATIONS OF FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR) IN PROTEOMICS

Proteomics [89,90] is the study of the entire complement of proteins expressed by a cell or tissue type. The focus of a proteomics experiment, for example, might be identiication of proteins that differ according to growth conditions or according to disease state. The aims are to identify and to characterize the maximum number of signiicant proteins. Proteomics experiments can be described as ‘bottom-up,’ in which proteins are digested with a protease and the resulting peptides are analyzed by mass spectrometry. Alternatively, a “top-down” approach, in which intact proteins are characterized, can be applied [53]. FT-ICR has found applications in both approaches, as discussed below.

5.4.1

‘BOTTOM-UP’ APPROACHES

‘Bottom-up’ proteomics involves digestion of proteins with a protease, usually trypsin, and subsequent mass spectrometric analysis of the resulting peptides. The masses of the tryptic peptides are characteristic of the parent protein. Either a peptide mass ingerprinting approach can be employed, or peptide sequencing by MS/MS can be performed. Peptide sequencing involves generally separation of the peptide mixture by on-line LC. As the peptides elute, they are ionized by electrospray and analyzed by MS/MS. In both the peptide mass ingerprinting and peptide sequencing methods, the data are searched against a protein database. 5.4.1.1 Peptide mass fingerprinting In proteomics, peptide mass ingerprinting of the peptide mixture is undertaken frequently by ionization by MALDI followed by time-of-light mass analysis. However, the high mass accuracy and resolving power of FT-ICR mass spectrometry can be exploited for this approach. It is possible to resolve virtually all peptide isobars differing by up to two amino acids, even those differing by the smallest mass difference of 3.4 mDa. Resolution of two peptides differing by 11 mDa in a complex mixture of 1000s of peptides has been demonstrated [91]. Clearly, isomers require MS/MS

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while, in the case of leucine/isoleucine, HECD is required. Further conidence in the accuracy of mass measurement can be gained through use of a dual electrospray source [92,93], in which an internal mass calibrant is introduced via a separate electrospray emitter. Consequently, space-charge effects in the ICR cell are mitigated without the inherent problem of electrospray ion suppression. Peptide mass ingerprinting can be performed by use of MALDI-FT-ICR [94,95]. For example, Przybylski and co-workers applied MALDI-FT-ICR to the proteomic analysis of cryoglobulins from a hepatitis C patient [96], and to alveolar proteomics associated with proteinosis and cystic ibrosis [97]. Alternatively, LC can be coupled with ESI FT-ICR for peptide mass ingerprinting [94]. Among other applications, LC coupled with ESI-FT-ICR has been used in the proteomic analysis of Escherichia coli [98], the proteomic analysis of amniotic luid [99], the identiication of brain natriuritic peptide (BNP-32) in plasma following heart failure [100], and in the molecular differentiation of ischemic and valvular heart disease [101]. An alternative approach, which exploits the high mass accuracy of FT-ICR, is the use of accurate mass tags (AMT) [17–19]. The approach involves initial creation of a set of AMTs which act as biomarkers for their parent proteins. Potential mass tags are generated by LC along with MS/MS performed on a conventional ion trap instrument, and then validated by FT-ICR and LC retention time. This initial procedure is relatively time-consuming but, once the AMTs are generated, high-throughput experiments can be performed subsequently. The approach has been applied to the global analysis of the Deinococcus radiodurans proteome [19], and proteomics analysis of breast carcinoma cells [102]. 5.4.1.2 Peptide Sequencing Methods incorporating FT-ICR MS/MS have been applied also to bottom-up proteomic analyes. Hakansson et al. [66] applied ESI FT-ICR and IRMPD MS/MS to the analysis of glycoproteins isolated from human cerebrospinal luid. Brock and co-workers [103] combined MALDI FT-ICR with SORI-CID. The throughput of this approach is hampered by the timescales associated with SORI-CID. Laskin and co-workers [104] compared approaches utilizing SORI-CID and SID coupled to ESI. The protein identiication scores were comparable for the two techniques. SID has the advantage that no pump-down delay is needed and, therefore, more cycles of MS/ MS can be completed. 5.4.1.2.1 Liquid Chromatography (LC) Tandem Mass Spectrometry (MS/MS) The majority of peptide-sequencing proteomic experiments involve coupling of LC with MS/MS. Protein spots may be excised from a two-dimensional (2-D) gel and digested prior to reversed-phase LC-MS/MS. Alternatively, a whole cell lysate may be digested and separated by 1 or 2-D (strong cation exchange and reversed phase) on-line LC followed by MS/MS; this approach is known as the shotgun approach [105]. The hybrid linear ion trap FT-ICR instrument allows simultaneous collection of MS data in the ICR cell and CID MS/MS data in the linear ion trap [106]. A common worklow involves one FT-ICR survey MS scan and linear ion trap CID scans of the three most abundant ions. Dynamic exclusion prevents re-analysis of precursor ions. The timescale for an FT-ICR scan is ca 1s (100,000 resolution at m/z 400) whereas a linear ion trap CID event takes ca 300 ms. This method is demonstrated in Figure 5.9.

141

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (a) #1345 RT: 29.86 512.2562

714.8323

489.5394 432.2817

707.6076

735.4080

524.9059 674.3078 570.7399 625.3184 400

500

600

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700 m/z

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(b) 513.16

#1346 RT: 29.87 Full ms2 524.91

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666.27 713.84

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(c) 974.16 #1347 RT: 29.88 Full ms2 625.32

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276.05 231.13 200

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300

606.00 547.98 530.07 500

851.28

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800

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1000

FIGURE 5.9 ‘Snapshot’ of an LC CID MS/MS analysis of a tryptic digest of a mixture of six proteins (bovine serum albumin, transferrin, cytochrome c, lysozyme, alcohol dehydrogenase, and β-galactosidase). RT = retention time. (a) FT-ICR survey scan (#1345); (b) Linear ion trap CID MS/MS of precursor m/z 524.9 (#1346); (c) Linear ion trap CID MS/MS of precursor m/z 625.3 (#1347); and (d) Linear ion trap CID MS/MS of precursor m/z 707.6 (#1348).

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Figure 5.9 shows a snapshot of an LC CID MS/MS analysis of a tryptic digest of a standard six-protein mixture. The top mass spectrum (scan # 1345 in this experiment) is an FT-ICR survey scan. In the subsequent scan (# 1346), CID MS/MS of the precursor ion m/z 524.9 is performed in the linear ion trap. That precursor ion is the most abundant ion in the survey scan that has not been subjected previously to MS/MS, that is, is not on the exclusion list. Scan # 1347 is the CID mass spectrum of precursor m/z 625.3, the second most abundant ion, not on the exclusion list, in the survey scan. The sequence ends with CID of precursor m/z 707.6, the third most abundant ion, not on the exclusion list, in the survey scan. The subsequent scan (not shown) is an FT-ICR survey scan. An alternative worklow favored by some researchers is one FT-ICR survey MS scan followed by CID in the linear ion trap of the 10 most-abundant ions [107]. These ‘parallel-processing’ approaches have been applied to a diverse range of studies including analysis of the chicken egg white proteome [108], the low molecular weight proteome of Halobacterium salinarum [109], the endocervical mucas proteome [110], sumoylation in Saccharomyces cerevisiae [111], and the tear luid proteome [112]. It is also possible to combine on-line LC with ECD MS/MS. This approach was applied to the analysis of the protein Fc-ROR2 that was isolated from chondrocytes and digested with trypsin [113]. Analysis by LC ECD MS/MS cannot be undertaken in a parallel manner: ECD must take place in the ICR cell. The previous survey scan must, therefore, be completed prior to ECD. Consequently, the duty cycle is reduced. A further disadvantage of this approach is the inherent ineficiency of ECD (see above). It is necessary to accumulate more precursor ions for ECD, with a concomitant increase in experiment time. The accumulation time is of the order of seconds rather than the milliseconds required for accumulation for CID. Nevertheless, studies have shown that ECD results in longer peptide sequence tags than does CID, thus improving conidence in peptide assignment [114]. Approaches which combine LC with ECD and CID have been developed also and are discussed further below [115–118]. 5.4.1.2.2 Post-Translational Modification (PTM) Analysis Post-translational modiication (PTM) of proteins plays a vital role in many biological processes. For example, phosphorylation is a key event in many signaling cascades, ubiquitination targets proteins for degradation, and glycosylation is involved in cell–cell recognition. Identifying and characterizing modiied proteins is a major goal in proteomics. The ease with which this can be undertaken depends largely on the lability of the modiication and its stoichiometry. ‘Parallel-processing’ methods in which high resolution, high mass accuracy FT-ICR survey scans are combined with lower speciication CID scans (see above) have been applied to the study of ubiquitination and sumoylation of proteins [119–121]. Both of these modiications are relatively stable. ‘Parallel-processing’ methods have been applied also to global analyzes of the phosphoproteome [122], and hundreds of phosphoproteins have been identiied. The Ascore algorithm, developed by Gygi and co-workers [123], can be applied to these data to determine site localization conidence. Phosphorylation of serine and threonine (but less so phosphotyrosine) are particularly labile modiications. CID of peptides containing either phosphoserine or phosphothreonine tends to result in loss of phosphoric acid (H3PO4,−98 Da) at the expense of peptide backbone

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product ions. Observation of a peak corresponding to the neutral loss conirms the presence of the modiication but often precludes its localization. Global phopshoproteome analyzes can be performed also by use of electron transfer dissociation (ETD) [124,125]. These analyzes utilized either 3D quadrupolar ion trap or linear ion trap instruments and are beyond the scope of this chapter. Targeted ECD approaches have been developed that exploit the advantages of ECD, in particular the retention of labile modiications on peptide backbone fragments, while minimizing the disadvantage of time scale. Experiments are performed on a hybrid linear ion trap FT-ICR mass spectrometer. Neutral losstriggered ECD (NL-ECD) [118] uses observation of a neutral loss as a trigger for an ECD event. The most abundant multiply-charged ion identiied in the FT-ICR survey scan is subjected to CID in the linear ion trap in the subsequent scan. When a neutral loss peak, for example, H3PO4, −98 Da, is observed, the following scan will be ECD of the precursor ion (not the neutral loss peak). An alternative approach involving separate LC analyses has been developed. The irst LC experiment involves CID of the eluting peptides. The purpose of this analysis is phosphopeptide discovery based on mass of the precursor and any sequence product ions observed. The m/z ratios of the putative phosphopeptides are added to an inclusion list and an LC-ECD analysis is performed. In this experiment, only those ions on the inclusion list are interrogated by ECD. This approach has been applied to the analysis of the protein Sprouty2 [126]. Fourteen sites of phosphorylation were identiied of which 11 were novel. Zubarev and co-workers utilized a combined ECD CID approach for the bottom-up analysis of phosphorylation in human α-casein [127]. The method involved an FT-ICR survey scan followed by ECD and CID of the two most abundant precursor ions. These researchers identiied a site of phosphorylation that, although known in the bovine form, had not been reported previously for human α-casein.

5.4.2

‘TOP-DOWN’ APPROACHES

FT-ICR mass spectrometry has great potential for ‘top-down’ proteomics [128], that is, characterization of intact proteins. The high resolution and mass accuracy are well-suited to the analysis of large biomolecules. Moreover, these features allow direct and accurate mass measurement of multiply-charged product ions, that is, in top-down MS/MS. To date, top-down MS/MS has been applied to characterization of proteins and large polypeptides up to 60 kDa [129]. The top-down approach offers some advantages over the bottom-up approach for protein characterization. Because intact proteins are analyzed, 100% sequence coverage is achieved. The method is, therefore, particularly suited to PTM analysis. The highest mass molecule for which unit resolution was achieved was 112,508 Da [20] by use of a 9.4 T instrument. As FT-ICR magnetic ield strength increases, unit resolution should be achieved for even higher mass molecules. Unit resolution enables modiications such as disulide bridge formation (−2 Da) or deamination ( + 1 Da) to be identiied. A disadvantage of the bottom-up approach is that any connectivity between modiications or mutations in the protein sequence is lost. For example, a protein with

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mutation at amino acid Xxx may be phosphorylated uniquely at amino acid Zzz. Any such information is lost following proteolytic digestion. For example, Ge et al. [130], performed top-down analyses of proteins isolated from Mycobacterium tuberculosis. One protein assignment, which was based on the mass of the intact species, was found subsequently to be erroneous based on ECD MS/MS data. The MS/MS results showed that, in fact, the species was a truncated version of a different protein. It would not have been possible to demonstrate this distinction using a bottom-up approach. A potential drawback for top-down analyses is that, as the molecular weight of a protein increases, it becomes less likely that the monoisotopic peak will be observed. For proteins ≥ 15 kDa, the monoisotopic peak is  0.9 and az between –0.3 and –0.8, the kinetic energy imparted to the ion by the ield is so high that ion behavior in the proximity of the stability boundary differs from that expected on the basis of theoretical calculations due to resonant excitation/ejection. For instance, in the case of 3 (Figure 12.10), ions were ejected due to trajectory instability at the working point az = –0.500 and qz = 0.938, calculated from Equations 12.2 and 12.1, respectively. Thus the working point (0.500, 0.938) deines this point on the experimentally-determined βz = 1 boundary of the stability diagram. The qz -value of the computed βz = 1 boundary of the stability diagram at az = –0.500 is 0.958. Thus, at az = –0.500, the difference in qz -values between the experimentally-determined βz = 1 boundary of the stability diagram and the computed βz = 1 boundary of the stability diagram is 0.020. When 1 is analyzed (Figure 12.8), ions were ejected due to trajectory instability at the working point az = –0.520 and qz = 0.900, calculated again from Equations 12.2 and 12.1, respectively. Thus the working point (0.520, 0.902) deines this point on the experimentally-determined βz = 1 boundary of the stability diagram. The qz -value of the computed βz = 1 boundary of the stability diagram at az = –0.520 is 0.966. Thus, at az = –0.520, the difference in qz -values between the experimentallydetermined βz = 1 boundary of the stability diagram and the computed βz = 1 boundary of the stability diagram is 0.064. In fact, regardless of the duration of the cooling time, the application of a DC component brings ions into a region of the ion trap in which the RF ield is larger and the ions are accelerated to higher kinetic energies. Because of this effect, ions undergo energetic collisions with the background gas and depletion of the monoisotopic ion signal intensity is observed due to unwanted fragmentation. Closer examination of the product ion mass spectra indicates the presence of product ions produced by collisions with the buffer gas. The results suggest that variation of the waveform duty cycle, which is achieved at the software level by entering different values in the scan table, can result in a relatively simple approach for generating BAD without the necessity of an additional power supply. The behavior of 1 can be explained considering the different charge state of the ions used as probes. Ion 1, which displays the largest discrepancy between the

383

Boundary-Activated Dissociation (BAD) in a Digital Ion Trap (DIT)

computed βz = 1 boundary and the experimentally-determined βz = 1 boundary, is a doubly-charged species, with the m/z-value close to those for 2 and 3. Its interaction with the trapping ield is consequently stronger: in other words, the doubly-protonated species is able to increase its kinetic energy at (az, qz)-values that are lower than those necessary to excite similarly 2 and 3 to achieve a comparable enhancement of kinetic energy. If this hypothesis is true, the loss of ion 1 in the right-hand side of the stability diagram is not due to its ejection from the ion trap and/or its discharge on the trap walls, rather it is caused by the activation of decomposition channels due to the increase of kinetic energy and effective collisions with helium. To verify this hypothesis, some experiments were performed by using different (az, qz)-values, corresponding to the points (a)–(d) shown in Figure 12.13. The mass spectra obtained under these conditions are shown in Figure 12.14. Singly-charged ions, at m/z-values higher than the doubly-charged precursor ion, are observed, providing evidence on the occurrence of boundary-activated chargeseparation dissociations. Among all the various experimental conditions examined, those leading to the best result (with respect to signal-to-noise ratio) correspond to point (b), which is reasonable considering the larger qz -range available that permits the storage of high-mass ions. However, when loci (discussed below) are drawn from the origin through the working points (a)–(d), it is found that the lengths of the loci lying within the stability diagram decrease in the order (a)–(d). Inspection of Figure 12.13 yields the qz-values for the intersection of these loci with the βz = 0 boundary from which the highest m/z-value for product ions remaining conined in the DIT can be calculated. The product ion mass/charge ratio reaches its greatest value at m/z 1730 for (a); m/z 1200 for (b); m/z 765 for (c); and m/z 672 for (d). As shown by the values for the high-mass/charge limit for points (a)–(d), m/z 555 is in no danger of being ejected and so it is not surprising that its intensity is sensibly constant. For the 0.2 0.1 0 –0.1

az

–0.2

0

0.1

0.2

0.3

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0.5

0.6

0.7

0.8 a 0.9

1

11

qz

b

–0.3 –0.4 –0.5

c d

–0.6 –0.7 –0.8

FIGURE 12.13 Selected working points employed to perform BAD experiments on [M+2H]2+ ions of bradykinin; (a) (–0.140, 0.750); (b) (–0.260, 0.815); (c) (–0.450, 0.895); and (d) (–0.570, 0.926).

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Normalized accumulated intensity

(a) 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5

[M+2H]2+ b5

515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559

Mass/charge Normalized accumulated intensity

(b) 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5

b5

515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559

Mass/charge

Normalized accumulated intensity

(c) 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5

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515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559

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b5

515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559

Mass/charge

FIGURE 12.14 Product ion mass spectra obtained using BAD of [M+2H]2+ of bradykinin at the following selected working points identiied in Figure 12.13: (a) (–0.140, 0.750); (b) (–0.260, 0.815); (c) (–0.450, 0.895); and (d) (–0.570, 0.926).

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385

point (d), the loss of some high-mass ions is expected. The locus of (az, qz)-values for all product ions is a straight line connecting point (d) (in Figure 12.13) to the origin of the stability diagram; it is seen that part of this locus falls outside the βz = 0 boundary of the stability diagram such that the trajectories of a range of product ions are rendered unstable and the ions are lost. These data indicate that BAD implemented in the DIT represents a realistic alternative for MS/MS experiments.

12.3

CONCLUSION

Among all non-resonant activation techniques, BAD has been shown to have unique advantages for the formation of product ions. Due to the necessity to utilize an additional power supply for generating the DC component, such an approach has not been used in any commercial mass spectrometer. Conversely, in the DIT, variation of the duty cycle of the rectangular waveform is controlled at software level and it allows readily introduction of the DC component for BAD experiments.

REFERENCES 1. Fulford, J.E.; Hoa, D-N.; Hughes, R.J.; March, R.E.; Bonner, R.F.; Wong, G.J. Radiofrequency mass selective excitation and resonant ejection of ions in a three-dimensional quadrupole ion trap. J. Vac. Sci. Technol. 1980, 17, 829–835. 2. Louris, J.N.; Cooks, R.G.; Syka, J.E.P.; Kelley, P.E.; Stafford Jr., G.C.; Todd, J.F.J. Instrumentation, applications and energy deposition in quadrupole ion trap MS/MS spectrometry. Anal. Chem. 1987, 59, 1677–1685. 3. Gronowska, J.; Paradisi, C.; Traldi, P.; Vettori, U. A study of relevant parameters in collisional-activation of ions in the ion-trap mass spectrometer. Rapid Commun. Mass Spectrom. 1990, 4, 306–314. 4. Louris, J.N.; Brodbelt, J.E.; Cooks, R.G. Photodissociation in a quadrupole ion trap mass spectrometer using a iber optic interface. Int. J. Mass Spectrom. Ion Processes 1987, 75, 345–352. 5. Lifshitz, C. Dissociative photoionization in the vacuum UV region with ion trapping. Int. J. Mass Spectrom. Ion Processes 1991, 106, 159–173. 6. McLuckey, S.A.; Goeringer, D.E.; Glish, G.L. Collisional activation with random noise in ion trap mass spectrometry. Anal. Chem. 1992, 64, 1455–1460. 7. Julian, R.K.; Cox, K.; Cooks, R.G. Proc. 40th ASMS Conference on Mass Spectrometry and Allied Topics. Washington, DC, 31 May–5 June 1992, p. 943. 8. Pannell, L.K.; Pu Q.L.; Mason, R.T.; Fales, H.M. Fragment pathway analysis using automated tandem mass spectrometry on an ion-trap mass spectrometer. Rapid Commun. Mass Spectrom. 1990, 4, 103–107. 9. Lammert, S.A.; Cooks, R.G. Pulsed axial activation in the ion trap: A new method for performing tandem mass spectroscopy (MS/MS). Rapid Commun. Mass Spectrom. 1992, 6, 528–530. 10. Varian, Walnut Creek, CA, USA, Technical literature. 11. Paradisi, C.; Todd, J.F.J.; Traldi, P.; Vettori, U. Boundary effects and collisional activation in a quadrupole ion trap. Org. Mass Spectrom. 1992, 27, 251–254. 12. Paradisi, C.; Todd, J.F.J.; Vettori, U. Comparison of collisional activation by the ‘boundary effect’ vs. ‘tickle’ excitation in an ion trap mass spectrometer. Org. Mass Spectrom. 1992, 27, 1210–1215.

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13. Traldi, P.; Catinella, S.; March, R.E.; Creaser, S.C. Boundary excitation. In Practical Aspects of Ion Trap Mass Spectrometry, eds. R.E. March and J.F.J. Todd, Vol. 1, Chapter 7, pp. 299–341. CRC Press, Boca Raton, 1995. 14. March, R.E.; Todd J.F.J. Quadrupole Ion Trap Mass Spectrometry. 2nd Edn. John Wiley & Sons, Hoboken, NJ, 2005, pp. 280–290 (references cited therein). 15. Vachet, R.W.; Glish, G.L. Boundary-activated dissociation of peptide ions in a quadrupole ion trap. Anal. Chem. 1998, 70, 340–346. 16. Ding, L.; Sudakov, M.; Kumashiro, S. A simulation study of the digital ion trap mass spectrometer. Int. J. Mass Spectrom. 2002, 221, 117–138. 17. Konenkov, N.V.; Sudakov, M.; Douglas, D.J. Matrix methods for the calculation of stability diagrams in quadrupole mass spectrometry. J. Am. Soc. Mass Spectrom. 2002, 13, 597–613. 18. Berton, A.; Traldi, P.; Ding, L.; Brancia, F.L. Mapping the stability diagram of a digital ion trap (DIT) mass spectrometer by varying the duty cycle of the trapping rectangular waveform. J. Am. Soc. Mass Spectrom. 2008, 19, 620–625.

Study of Ion/ 13 The Molecule Reactions at Ambient Pressure with Ion Mobility Spectrometry and Ion Mobility/Mass Spectrometry Gary A. Eiceman and John A. Stone CONTENTS 13.1 13.2

Introduction ................................................................................................ 388 The Ion Mobility Spectrometer and a Mobility Measurement ................... 389 13.2.1 The Proiles of Ion Mobility Spectra Used to Obtain Thermodynamic and Kinetic Data ............................................... 391 13.2.1.1 Type 1. Equilibrium A + + B = AB + Exists Throughout the Source and Drift Region .................... 393 13.2.1.2 Type 2. A + + B→AB + in the Drift Region .................. 393 13.2.1.3 Type 3. A+ and AB + are Formed in the Source Region and Neither A Nor B is Present in the Drift Region .......................................................................... 394 13.2.2 Examples Where Thermodynamic and Kinetic Data have been Obtained from Ion Mobility Spectra.................................... 394 13.2.2.1 Type 1. Ions in Equilibrium Showing a Single Peak in the Mobility Spectrum ............................................. 395 13.2.2.2 Type 2. Reaction Rate Constant Measurements ..........400 13.2.2.3 Type 3. Dissociation of Adduct Ions ............................403 13.2.3 The Kinetics of Thermal Electron Capture and Thermal Electron Detachment ....................................................................406 13.2.3.1 Electron Capture ..........................................................406 13.2.3.2 Thermal Electron Detachment.....................................409

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13.3 Concluding Remarks .................................................................................. 411 References .............................................................................................................. 412

13.1

INTRODUCTION

In the Prefaces to Volumes 4 and 5 of Practical Aspects of Trapped Ion Mass Spectrometry the Editors have explained that, in deining the scope of these publications, it is considered that “an ion is ‘trapped’ when its residence time within a deined spatial region exceeds that had the motion of the ion not been impeded in some way.” Ion mobility spectrometry (‘IMS’) operated at atmospheric pressure, which falls clearly within this deinition, involves the determination of the time taken for the components of a packet of ions to move through a deined distance under the inluence of a speciied electric ield gradient and against the counter current low of a drift gas at ambient pressure. Ion mobility spectrometers have been utilized for monitoring hazardous or controlled substances in a range of venues on land, in light, in space, and underwater in submarines, and are ubiquitous at airports for detecting explosives in carry-on articles [1–18]. Such uses have arisen from the pragmatic attractions of these analyzers including ruggedness, small size, and affordability. Measurements by IMS are based on the production and determination of gaseous ions derived from a sample and are made commonly at ambient pressure so vacuum systems and associated pumps are unnecessary [19–22]. These features account for the portability of IMS analyzers and, along with the convenience of use and speed of operation, often make mobility spectrometers the instrument of choice for in-ield, routine, measurements. An early term for IMS was plasma chromatography from the presence of a plasma, that is, both positive and negative ions in the ion source, and from the separation of ions in the supporting medium, air, or nitrogen. As with gas or liquid chromatograms, ion mobility spectra alone lack the facility for unequivocal identiications because the relationship of an ion mobility measurement to the structure or identity of an ion is under-developed. Consequently, mass spectrometers were combined with mobility drift tubes early in the development of IMS to provide ion identities through mass analyses. The combination of mobility and mass measurements can also permit the study of reactions between gaseous ions and molecules at ambient pressure in air, or other gases, and the measurement of both kinetic and thermodynamic values. Thermodynamic data that are suitable for tabulation include standard enthalpies, entropies, and free energies and can be regarded as universally applicable for systems at speciied temperature when all participants are at thermal equilibrium. Though such data can also be obtained without thermal equilibrium, compensating experiments, or mathematical corrections are required, sometimes creating dificulties in practice and/or interpretation. A chemical system in the gas phase can reach thermal equilibrium, at a deined temperature, when a suficient number of intermolecular collisions produce a Boltzmann distribution of energies in all modes, electronic, vibrational, rotational, and translational. In measurements made with an ion trap instrument or Fourier Transform Ion Cyclotron Resonance (FT-ICR) spectrometer at low pressure, hot ions must be cooled, commonly with a pulse of buffer

The Study of Ion/Molecule Reactions at Ambient Pressure

389

gas, and time allowed for thermalization, with the number of thermalizing collisions directly proportional to pressure. Such techniques are unnecessary with an ion mobility spectrometer at ambient pressure because each ion experiences more than 1010 collisions per second, mainly with neutral atoms or molecules of the supporting gas atmosphere. When the concentration of a sample compound is 1 ppm by volume, for example, then, on an average, an ion undergoes one collision with a molecule of the sample compound for every million collisions with the molecules of the gas atmosphere. Thus, there are ca 104 ion/sample-molecule collisions per second for possible reactions, under thermalized conditions for well-deined temperatures. Additionally, the residence time of an ion in an ion mobility spectrometer operating at atmospheric pressure is ca 5–50 ms, which allows the study of the interactions of ions with molecules at very low concentrations. A further advantage with IMSbased thermochemical determinations is that the available temperature range, from sub-ambient to more than 500 K, is far greater than that available with many other experimental methods. In spite of the advantages cited above, ion mobility spectrometers operating at atmospheric pressure have been used infrequently to obtain physical chemical data, kinetic and thermodynamic, in the study of ion/molecule chemistry. In this chapter, an overview is given on the type of information obtainable from ion mobility studies at atmospheric pressure and the variety of experimental methods employed in such studies. The data obtained under well-deined conditions agree favorably with those from other more frequently used methods, for example: (i) pulsed high pressure mass spectrometry (PHPMS), which is operated at well-deined temperatures but at pressures ca 200 times lower than IMS; and (ii) FT-ICR and ion trap mass spectrometers, which are operated under vacuum.

13.2

THE ION MOBILITY SPECTROMETER AND A MOBILITY MEASUREMENT

An ion mobility spectrometer is a simple device with three essential elements: a source region, a drift region, and a detector. The drift tube usually is cylindrical with an overall length from 5 to 10 cm and an internal diameter from 1 to 5 cm. Metal rings separated by insulating material, for example Telon®, provide a uniform electrostatic drift ield when the source end is at high potential and the detector is essentially at ground potential. The high potential is positive for the detection of positive ions and negative for negative ions. The source region is separated from the drift region, as shown in Figure 13.1, by an ion shutter composed of two closelyspaced sets of interdigitated wires, grids 1 and 2 in Figure 13.1 [23]. When the grids are set at the same potential and consistent with their position in the spectrometer, ions pass unhindered from source to drift region; here the ion shutter is open. An imposed potential difference of ca 50–100 V between the two closely-spaced grids creates an electrostatic ield far greater than the drift ield that is typically around 220 V cm–1, so that ions are drawn to the grid wires and discharged: the ion shutter is now closed. Ions are formed commonly in the source region using a radioactive nickel foil, though other sources, including ultra violet (UV) discharge lamps and corona discharge, have been described. Ions are gated into the drift region in

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S

M 63Ni

I

D

G1 G2 F A

T

P

B

H

FIGURE 13.1 Schematic diagram of an ion mobility spectrometer: A, ampliier; B, metal shell; D, drift gas inlets; F, Faraday plate; G1, G2, grids constituting the ion shutter; H, heating tape; I, insulation rings; M, metal ield rings; P coils for pre-heating sample and drift gases; S, sample gas inlet; and T, threaded support rod.

pulses of 30–300 µs-duration by opening and closing the ion shutter at frequencies of 20–100 Hz. The ions move under the inluence of the uniform electrostatic ield to the detector, a Faraday plate, where signal is generated. The ampliied signal, displayed as a function of the time of arrival at the detector, that is drift time, constitutes the ion mobility spectrum. The separation of ions into individual swarms occurs according to their differences in mobilities as swarms drift from the ion shutter to the detector. The drift gas is usually either puriied air or nitrogen and the low (300–1000 cm3 min–1) of the drift gas is from the detector to the ion shutter, that is, counter to the ion drift direction. This gas low is mixed with the sample-containing source gas (10–100 cm3 min–1) before exiting the instrument at the source end, so the entry of sample vapors into the drift region is suppressed in this coniguration of gas lows. The time of drift (td) for an ion swarm in the drift region (of length, l) yields a drift velocity vd given by vd =

l td

(13.1).

The drift velocity, when normalized for electric ield strength, E, produces the mobility coeficient, K, as shown in Equation 13.2 vd = KE

(13.2).

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391

Because vd and hence K are temperature and pressure-dependent, values for K are usually normalized to 273 K and 760 mm Hg and are reported as the reduced mobility coeficient, Ko,  273   P  Ko = K   T   760 

(13.3),

where T is temperature in kelvin and P is pressure in mm Hg. If the character of the ion does not change Ko has a constant value but, upon change of temperature, differences in ion hydration or clustering with other ambient molecules result in nonconstant Ko values. An ion under the inluence of the electric ield in the drift region acquires kinetic energy, some of which is lost by collision to the surrounding gas molecules. When an ion is to be accepted as thermal, it is important that the retained kinetic energy (1/2 mvd2 + 1/2 Mvd2 ) is not signiicant compared with thermal kinetic energy (3/2kbT ) as found in the average kinetic energy of an ion (KEav), which is approximated by the Wannier expression: KEav =

3 1 1 kbT + mvd2 + Mvd2 2 2 2

(13.4),

where m is the mass of the ion, M is the mass of the drift gas molecule [24]. A calculation of these energies is illustrated for protonated 2,3-dimethyl pyridine with a drift velocity of 769 cm s–1 in a ield of 280 V cm–1 at 350 K and 660 mm Hg of N2; the thermal energy term for the drift gas 3/2 kbT has the value 7.3 × 10 –21 J and the retained kinetic energy 1/2mvd2 + 1/2 Mvd2 has the negligible value of 6.8 × 10 –26 J. When this condition holds, a mobility spectrometer is said to be operating in the socalled low-ield region and ions are regarded as thermalized. The low-ield region is assumed usually when E/N is less than 2 Td, where E is the electric ield (V cm–1), N is the number density of molecules (cm–3), and Td is the townsend (10 –17 V cm2). The low-ield region is accessed readily with ion mobility spectrometers operating at atmospheric pressure but it is dificult to achieve satisfactorily with instruments that operate at pressures of ca 1 mm Hg. In the low-ield region, the mobility coeficient at a ixed temperature is independent of ield strength, which can be 100–600 V cm–1 [25]. The lower limit for E is determined practically by radial losses of ions to the walls of the drift tube; the upper limit is deined by electrical breakdown of gases in the supporting atmosphere inside the drift tube. An important property of an ion mobility spectrometer operating in the low-ield region is that ion losses to the walls by radial diffusion do not introduce mass discrimination in the collected ion signal because the ratio of the radial spreading distance to the drift distance is independent of ion mass [24].

13.2.1

THE PROFILES OF ION MOBILITY SPECTRA USED TO OBTAIN THERMODYNAMIC AND KINETIC DATA

There are two approaches that may be taken to follow the course of an ion/molecule reaction occurring in an ion mobility spectrometer. The irst is through the proile of the ion mobility spectrum alone, and the second is from mass spectral ion intensities.

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When possible, the irst method is preferable because there is always the danger of mass discrimination, ion/neutral association, or collisional dissociation when ions pass from the high pressure of a mobility spectrometer to the vacuum of a mass spectrometer. Despite these complications, mass spectrometry has value for identifying ions because otherwise ion identity must be deduced intuitively by reference to known or anticipated reactions and ion behavior in a mobility drift tube. A single type of ion, that remains unaltered during the period following injection into the drift region to its arrival at the detector, produces a near-Gaussian peak in the ion mobility spectrum. The width of the peak is determined by a combination of the pulse width of the ion shutter and by Brownian motion [26]. A slight asymmetry in the peak arises from the increased axial diffusion time experienced by the late-arriving ions. When two ions formed in the source transit the drift region with no further change, two peaks are observed, as illustrated in Figure 13.2a for ions A+  and B + . Such spectra cannot provide quantitative physical chemical data because conditions in the ion source, such as concentration gradient of neutral molecules, electrostatic ield, and reaction time, are not well deined. Early attempts to use this method to obtain proton afinity differences from the ratio of the intensities of protonated polycyclic aromatic hydrocarbons yielded only the order of proton afinities [27]. When a process occurring in the drift region relates the ions, the mobility spectrum may become either simpler or more complex than shown in Figure 13.2a. Nonetheless, when experimental conditions are controlled, the spectrum may be interpretable and, subsequently, may provide thermodynamic and/or kinetic information pertinent to the process or ion/molecule chemistry. Consider the simple (b) Relative intensity

Ion intensity

(a) A+ B+

8

10

12

14

16

18

20

22

24

1.2 Type 1 1 0.8 0.6 0.4 0.2 0 8

A+and AB+

18

Drift time (ms) 1.2 Type 2 1 A+ 0.8 0.6 0.4 0.2 0 8

(d) Type 3

AB+

13

18

Ion intensity

Relative intensity

(c)

A+ AB+ 8

10

12 14 16 18 Drift time (ms)

20

22

FIGURE 13.2 Schematic mobility spectra illustrative of ion/molecule processes: (a) Ions A+  and B+ are formed in the source and experience no reaction in the drift region; (b) equilibrium A +  + B = AB+ prevails throughout the drift region; (c) A +  + B → AB + in the drift region; and (d) AB + →A +  + B in the drift region.

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393

association reaction described by Equation 13.5, in which the ions have a single positive charge (this discussion applies equally to negatively-charged ions) A + + B = AB+

(13.5).

At atmospheric pressure, third-body stabilization of AB + is highly eficient and the reaction may be treated as second-order in the forward direction and irst-order in the reverse direction. In addition, the concentration of B is usually much greater than the concentration of A+ , so that the reaction is pseudo-irst order in the forward direction with a constant concentration of B. As detailed below, ion/molecule reactions or interactions may be investigated using several experimental designs, each of which has been demonstrated with speciic features or advantages. The ion mobility spectra produced in these experimental conigurations differ and will be designated Types 1, 2, and 3. Type 1. Equilibrium A +  + B = AB + Exists Throughout the Source and Drift Region In the Type 1 experiment, both A + and AB + are formed in the source region and the concentration of B is uniform throughout both source and drift regions. When the concentration of B is much greater than that of the ions, the reaction will attain equilibrium prior to the ions reaching the shutter and equilibrium will prevail in the drift region. The charge spends part of the time on A and part of the time on AB so there is only one peak in the mobility spectrum (Figure 13.2b) and its arrival time (time of maximum ion intensity) depends on the relative equilibrium concentrations of A+ and AB + . The arrival time for this composite peak is intermediate between the arrival times of A+ and AB + without interactions (Figure 13.2a) and is the weighted ion number average of the ion mobilities, which is expressed by Equation 13.6 in terms of the ion mole fractions Xi and their individual arrival times ti

13.2.1.1

td = X A+ t A+ + X AB+ t AB+

(13.6).

13.2.1.2 Type 2. A + + B → AB + in the Drift Region In this category, A + ions and some AB + ions are formed in the source region, B is present at uniform concentration in the drift region, and the reaction has not gone to completion (that is, attained equilibrium) when ions are introduced into the drift region. Discrete peaks at t A+ and t AB+ are present for A + and AB + that pass unchanged through the drift region. Some AB + ions are formed as A + travels to the detector, the rate of the association reaction being given by: −

d[A + ] = k[ A + ][ B] dt

(13.7),

where k is the rate constant. The rate of reaction is greatest near the shutter where the concentration of A + is highest, and AB+ ions formed here will arrive at the detector at a time close to t AB+ .

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

The rate of reaction is least near the detector, where the concentration of A+ is the smallest, and ions AB + formed here will have spent most of their transit time as A + and will arrive at times close to t A+ . The ‘ill in’ intensity between the normal peaks for A + and AB + is lowest at t A+ and rises exponentially to t AB+ , as illustrated by Figure 13.2c. The same type of mobility spectrum, as is shown in Figure 13.2c for the association reaction, Equation 13.5, is observed when the reaction is a charge exchange, as in Equation 13.8, where B is again present at uniform concentration throughout the drift region A + + B → A + B+

(13.8).

Type 3. A + and AB + are Formed in the Source Region and Neither A Nor B is Present in the Drift Region The dissociation of AB + , formed in the source region and injected into the drift region, will be observable at a temperature consistent with the activation energy for dissociation; the higher the activation energy, the higher is the required temperature. Signiicant reaction in this irst-order process can be observed when the rate constant is of the order of the reciprocal of the drift time, which is usually of ca 100 s–1; however, the drift time can be varied to a limited extent by changing the electrostatic ield. The mobility spectrum will show two distinct peaks, at time t A+ for A + and at time t AB+ , the drift time for AB + without decomposition within the drift region. Ions arriving at the detector at intermediate times have spent the irst part of their drift time as AB + and the second part as A + . The rate of reaction, given by Equation 13.9, is greatest at the entrance to the drift region, where the concentration of AB + is highest, and least at the detector. The plot in Figure 13.2d illustrates the resulting exponential ‘ill in’ of the spectra between the two discrete peaks with maximum intensity at t A+ . 13.2.1.3

−

d [ AB+ ] = k[ AB+ ] dt

(13.9),

where k is the rate constant.

13.2.2

EXAMPLES WHERE THERMODYNAMIC AND KINETIC DATA HAVE BEEN OBTAINED FROM ION MOBILITY SPECTRA

Though IMS and ion mobility spectrometry/mass spectrometry (IMS/MS) methods may not be recognized widely for determining values for enthalpy, entropy, and kinetic constants, signiicant experience in the study of reactions at ambient or elevated pressures exists. In the discussion below, examples are drawn from gas-phase reactions for associations and displacements of ions using either combined mobilitymass spectrometry or, in some instances when the chemistry was well known, a drift tube alone. The order of presentation follows that used in the prior section. In a later discussion, reactions with electrons are described.

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395

13.2.2.1

Type 1. Ions in Equilibrium Showing a Single Peak in the Mobility Spectrum In dried air or nitrogen with ionization using a 63Ni source, the initial ionization event produces N + , N2+ , O + , and O2+ which lead, through a series of rapid ion/molecule reactions with the ubiquitous trace amounts of water, to the hydronium ion (H2O)nH + [20]. The subscript n denotes a range of values that depends on both the water concentration and the temperature. The equilibrium of the hydronium ion is shown in Equation 13.10, (H 2O)n H + + H 2O = (H 2O)n+1H +

(13.10).

For example, using data from reference [28] and assuming equilibrium conditions with 1.0 ppmv (parts per million by volume) water at 300 K, the populations for n = 2, 3, 4, and 5 are 0, 21, 76, and 3%, respectively. At 400 K, the respective populations are 47, 53, 0.1, and 0%. Proton transfer from one or more of these hydrates to a molecule, present at a concentration much lower than that of water, which is present usually at a concentration of 1–10 ppmv, is often the initial step in forming an ion of interest. Though different experimental methods have been employed in the study of physical chemistry of the proton hydrate, it is not surprising that investigations have been made also with IMS given the importance of the hydronium ion as a reactant in IMS. An early attempt was made by Kim et al. to link the reduced mobility of the hydrated proton to the known range of n for (H2O)nH + in moist atmospheric air [29]. An ion mobility spectrometer interfaced to a mass spectrometer was used with a constant concentration of water throughout the drift tube of the mobility spectrometer, thereby ensuring a constant ratio of the equilibrium concentrations of the various hydrates in the drift region. In the absence of sample molecules, the hydrates constitute the major peak in the mobility spectrum, the reactant ion peak (RIP), whose drift time varies as the value of n changes with change of temperature. An ion/molecule association reaction is always exothermic, so that raising the temperature favors smaller clusters and vice versa. A larger cluster ion has lower mobility than does a smaller one, and so the drift time of the RIP decreases with increase of temperature while a lower temperature leads to a longer drift time. Each hydrate ion has a unique mobility and the drift time of the single peak in the spectrum should give a measure of the equilibrium distribution. The mobility, and hence the reduced mobility, increased as the temperature was raised, consistent with a reduction in the average value of n as the distribution shifted to the lighter, more mobile hydrates. The water concentration was not measured but was calculated by Equation 13.11 from the mass spectral intensities In and In–1 (presumably at one temperature, although this is not stated) together with the equilibrium constants Kn–1,n from the thermodynamic data of Kebarle et al. [30]. PH2O =

In I n−1K n−1,n

(13.11).

The distribution diagram for the proton hydrates was calculated over the whole temperature range with this water concentration and the equilibrium constants. The

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measured ion mobility over the whole temperature range is in good agreement with that obtained from the formula in Equation 13.12, which relates the mobility coeficient K to the ion/molecule interaction potential Ω, the measure of closest approach of ion and molecule rm, and their reduced mass µ [1], K=

3  2π  16 N  µ kbT 

1/2

1 π rm2Ω

(13.12).

The same studies were reported for the hydrates of NO + and NH4 + , which are minor ions in the same system. When equilibrium exists in an ion/molecule reaction then the relative peak heights of the different hydrate ions, sampled from a mobility spectrometer through a small oriice into a mass spectrometer, give a direct measure of their equilibrium concentrations, provided there is no mass discrimination in the sampling process. Gheno and Fitaire applied this method to obtain thermodynamic data for the proton hydrate equilibria over the temperature range 300–473 K with N2 drift gas containing 3 ppmv water [31]. Van’t Hoff plots yielded the standard enthalpy and entropy values of Table 13.1 that show excellent agreement with National Institute of Standards and Technology (NIST) values [28] of both the enthalpy (–ΔrHo) and entropy (–ΔrSo) changes for the formation of (H2O)3H + but less so for (H2O)4H + . The reduced agreement with NIST values for (H2O)4H + is due probably to dissociation in the interface to the mass spectrometer of the higher, more fragile n = 4 hydrate. The enthalpy change for the association of N2 with NO + and to a lesser extent the entropy change are in good agreement with the values obtained by PHPMS at ca 4 mm Hg [32]. The data for such weakly-bound clusters as N2·NO + must always be treated with some skepticism because there is usually a danger that the ions detected are clustered with drift gas molecules during adiabatic cooling in the free-jet expansion between the mobility drift tube and the mass spectrometer [33]. However, it would be surprising if exactly the same relative ion intensities in the mass spectrum occurred in expansion from atmosphere pressure and also from 4 mm Hg, and the equality of the thermodynamic data for the formation of the N2 · NO + complex validates the results, the IMS data, and the method. Preston and Rajadhyax who employed a hand-held ion mobility spectrometer, similar to those used in military applications, studied the correlation between the reduced mobility for ion processes at equilibrium and the arrival time of the single peak in the mobility spectrum [34]. Equilibrium constants were determined as a function of temperature for the formation of proton-bound dimers MZH + as shown in Equation 13.13, where each of M and Z can be pyridine, 3-(3-methoxypropoxy) propanol (DPM), acetone, or water, MH + + Z = MZH +

(13.13).

At equilibrium, the arrival or drift time td of the single peak is the number-weighted average of the drift times of the two constituent ions as described by Equation 13.6. When the times spent as the individual ions are t MH+ and t MZH+ , respectively, it follows that

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The Study of Ion/Molecule Reactions at Ambient Pressure

TABLE 13.1 Thermodynamic Data for Hydration Reactions from IMS/MS Determinations Reaction (H2O)2H +  + H2O = (H2O)3H +  (H2O)3H +  + H2O = (H2O)4H +  NO +  + N2 = N2 · NO + 

–ΔrHo (kJ mol−1)

–ΔrSo (J K−1 mol−1)

86 ± 4 (84 ± 5)a 58 ± 5 (73 ± 4)a 20 ± 4 (19 ± 1)b

109 ± 17 (94 ± 20)a 78 ± 17 (118 ± 8)a 58 ± 13 (71)b

Source: Gheno, F.; Fitaire, M. J. Chem. Phys. 1987, 87, 953–958. Evaluated data in reference NIST. NIST Chemistry Webbook. 1998. b PHPMS data from reference Hiraoka, K.; Yamabe, S. J. Chem. Phys. 1989, 90, 3268–3273. a

X MH+ a = t MH+ /td

and

X MZH+ = t MZH+ /td

(13.14),

and the measured reduced mobility for a peak at any temperature is related to the individual reduced mobilities by K o = X MH+ K o MH+ + X MZH+ K o MZH+

(13.15).

When Equations 13.14 and 13.15 are combined, the equilibrium constant is given by Equation 13.16 in which Po is the standard pressure, 101 kPa, and PZ is the partial pressure of Z K=

X MZH+ Po K MH+ − K o Po ⋅ ⋅ = o X MH+ PZ K o − K o MZH+ PZ

(13.16).

As the concentration of the neutral molecule Z was increased, the reduced mobility attained a limiting minimum value, calculated from the arrival time of the single peak. This value was taken to be K o MZH + , the concentration of MH + presumably being negligibly small. The term K o MH + was the reduced mobility in the absence of Z, and equilibrium constant measurements over a range of temperature yielded the results presented in Table 13.2. The standard enthalpy changes for acetone–water and pyridine–pyridine are in fair agreement with literature values but there is no agreement for pyridine–water. Some of the standard entropy changes are impossibly high and show little agreement with literature values. Although the method is sound, the instrument used for this study was not suited particularly to the determination of reliable thermodynamic data because the drift region was only 3.7 cm long. Giles and Grimsrud described an instrument designed speciically for the study of ion/molecule reactions [35]. The cylindrical drift tube was large, 40 cm long and 9 cm in diameter, and the moveable ion source allowed facile change in drift length.

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

TABLE 13.2 Thermodynamic Data for the Reaction MH +  + Z = MZH + as Measured by Mobility Spectrometry M Acetone Pyridine Pyridine DPM

Z

–ΔrHo (kJ mol–1)

–ΔrSo (J K–1 mol–1)

Water Pyridine Water Acetone

99±10 (84) 139±25 (103) 44±9 (97) 67±7

226±50 (109) 260±80 (124) 50±50 (116) 45±6

Source: Preston, J.M.; Rajadhyax, L. Anal. Chem. 1988, 60, 31–34. Note: Values in brackets are PHPMS data from reference NIST. NIST Chemistry Webbook. 1998.

Ion mobility spectra were recorded using a Faraday plate with a small central oriice that permitted passage of ions for identiication by a quadrupole mass spectrometer. Ion/molecule reaction rate constants and reaction enthalpies and entropies were determined using this IMS/MS instrument; one application followed the pioneering work of Preston and Rajadhyax [34] to measure the equilibrium constant for the association of CHCl3 and Clˉ. In this work, ions were formed in the source region and passed through a counter low of CHCl3 in the drift region. The arrival time of the single peak, initially due to Clˉ, increased with increasing concentration of chloroform, suggesting the formation of Clˉ(CHCl3), as in Equation 13.17: Cl − + CHCl 3 = Cl − (CHCl 3 )

(13.17).

The identity of this adduct ion was conirmed by the mass spectrum that contained both Clˉ and Clˉ(CHCl3). A limiting arrival time of the peak deined the concentration of CHCl3 at which essentially all the Clˉ was found in the adduct ion. When X Cl − and X Cl − ( CHCl3 ) are the equilibrium mole fractions of the two ions and tobs is the arrival time of the composite peak, which changes from to for Cl– to t1 for Clˉ(CHCl3), then: X Cl − + X Cl − (CHCl3 ) = 1

(13.18)

X Cl − to + X Cl − ( CHCl3 ) t1 = tobs

(13.19)

X Cl − ( CHCl3 ) X Cl − K=

=

tobs − to [Cl − (CHCl 3 )] = [Cl − ] t1 − tobs

1 tobs − to ⋅ t1 − tobs [CHCl 3 ]

(13.20)

(13.21)

The Study of Ion/Molecule Reactions at Ambient Pressure

399

so that 1 1 1 = + tobs − to K (t1 − to )[CHCl 3 ] t1 − to

(13.22).

A plot of (tobs–to) –1 vs [CHCl3]–1 yields a straight line with slope [K(t1–to)]–1. The mobility coeficient was determined at three different temperatures and ΔrHo = –75.7 kJ mol–1 and ΔrSo = –91.2 J K–1 mol–1 were obtained from the van’t Hoff plot; these values are in good agreement with listed values that range from –75.7 to –81.6 kJ mol–1 and –61.9 to –103 J K–1 mol–1, respectively [28]. An experiment with Brˉ forming Brˉ·CHCl3 found ΔrHo = –66.1 kJ mol–1 and ΔrSo = –88.2 J K–1 mol–1, the former value being appropriately smaller than for the Clˉ analog. The chloride anion is known as a useful reactant ion for IMS, particularly in the detection of explosives [1,2,36,37], as it forms complexes with molecules by bonding via hydrogen(s) rendered acidic by the presence of NO2 groups. Such complexes were not studied for explosives that in general have very low volatility, but rather for compounds of high volatility that are added in low concentration as chemical taggants. Information on the relative strength of binding between Clˉ and several potential taggants, 1,4-dinitrobenzene (DNB), 2,3-dimethyl-2,3-dinitrobutane (DMNB), and 2,3-dimethyl-2,4-dinitropentane (DMDNP) was obtained by Lawrence et al. from equilibrium measurements by IMS/MS [38]. The Clˉ reactant ion was formed in the source region by the incorporation of trace amounts of CH2Cl2 into the ultra high-purity nitrogen source gas; samples of each nitro compound (M) produced the Clˉ(M) adduct (Equation 13.23), which was suficiently stable to traverse the drift region to the detector: Cl − + M = Cl − (M)

(13.23).

A measure of the stability of each adduct was obtained by raising the temperature of the whole instrument to determine the highest temperature at which the adduct was observed in the mobility spectrum. The order of stability determined, Clˉ(DNB)  > Clˉ(DMDNP) > Clˉ(DMNB), is the same as the number of H atoms in a position α to the NO2 groups, that is, 4, 1, and 0. Further studies were made with DMNB because it has suitable properties, in particular vapor pressure, as a taggant. The standard enthalpy and entropy changes for the association of Clˉ with DMNB were determined by forming Clˉ in the source region and adding DMNB, in increasing and known concentrations, to the drift gas that lowed counter to the ions. A peak at time to when no DMNB was present was due to Clˉ. The single peak seen in the mobility spectrum with DMNB in the drift gas stream was identiied as a mixture of Clˉ and Clˉ (DMNB) using an IMS/MS instrument. When the DMNB concentration was increased further, the peak shifted to a longer drift time as shown in Figure 13.3. As concentration was increased further, the drift time attained a constant and maximum value, t1; the sole component of the peak was mass-identiied as Cl– (DMNB). Mass spectra for the composite peak at the lower concentrations of DMNB demonstrated the equilibrium of Equation 13.23. The interpretation of the results follows that of Giles and Grimsrud where a plot of (tobs –to) –1 vs [DMNB]–1 yielded a straight line with slope of [K(t1–to)]–1. The

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50 ppm DMNB

Signal intensity (arbitrary units)

9.3 ppm 2.7 ppm 0.7 ppm

0.2 ppm

0 ppm

5

10 15 Ion drift time (ms)

20

FIGURE 13.3 Ion mobility spectra as functions of the concentration of DMNB at 443 K. The peak at 0 ppm DMNB is the chloride ion alone and peaks with DMNB are due to the Clˉ(DMNB) adduct where drift time is dependent upon vapor concentration of DMNB in the drift tube. (Reproduced from Lawrence, A.H., et al., Int. J. Mass Spectrom. 2001, 209, 185–195. With permission from Elsevier.)

equilibrium constant K was determined at different temperatures and, from a van’t Hoff plot, ΔrHo = –92.1±3.1 kJ mol–1 and ΔrS o = –92.1±7.4 J K–1 mol–1 were obtained. 13.2.2.2 Type 2. Reaction Rate Constant Measurements Spectra of Type 2 were used for kinetic studies by Giles and Grimsrud who determined the rate constants for the SN2 displacement of Brˉ by Clˉ from methyl-, ethyl-, isopropyl-, and n-butyl-bromide at 398 K as per Equation 13.24 [35], Cl − + RBr → RCl + Br −

(13.24).

The chloride ion was formed by dissociative electron capture by CCl4 in a 63Ni ionization source; n-alkyl bromide, at a known concentration, was present in the drift region. The traces in Figure 13.4 are ion mobility spectra obtained at three concentrations of methyl bromide. Figure 13.4a shows the Clˉ peak in addition to two small peaks, each marked with an asterisk, that arise from impurities; the small

401

The Study of Ion/Molecule Reactions at Ambient Pressure 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0

(b)

0.06 0.04 0.03 0.02 0.01 0

(c)

(d)

Ion intensity (nA)

(a)

0.05 0.04 0.03 0.02 0.01 0 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 20

25

30

35

Drift time (ms)

FIGURE 13.4 Ion mobility spectra for the reaction of Clˉ with CH3Br at concentrations of (a) none, (b) 1.29 × 10l2, (c) 2.60 × 10I2, (d) 5.27 × 10l2 molecules cm–1. (Reproduced from Giles, K.; Grimsrud, E.P. J. Phys. Chem. 1992, 96, 6680–6687. With permission from the American Chemical Society.)

peaks were invariant with methyl bromide concentration and can be ignored. The intensity for Clˉ in the mobility spectra decreases as the Brˉ intensity increases with increasing concentration of CH3Br, as shown in Figure 13.4b through d. The contribution of each ion to the shape of the mobility spectrum was determined by concurrent mass spectrometry to obtain the relative mobility spectral area Ai assignable to each ion as a function of alkyl bromide (RBr) concentration. The pseudo-irst order reaction rate constant for reaction 13.24, k’ = k24[RBr], is given by Equation 13.25 in which td is the arrival time, that is the reaction time, of Clˉ:

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k′ =

1  ACl − + ABr −  ln  td  ACl −

(13.25).

The graph of k’ vs [RBr] is a straight line of slope k24 and the value for k24 was duplicated in a different experiment involving mass spectrometric detection with the drift tube acting as a reactor with the shutter open continuously. The intensities of Clˉ and Brˉ were measured as a function of RBr concentration with reaction time the same as for the pulsed mode; a plot of ln( I Cl − /I Cl − + I Br − ) vs [RBr] yielded a straight line of slope –k24td. The results obtained for the four alkyl bromides by the area method and the mass spectrometric method are compared in Table 13.3 with those obtained from a PHPMS study completed at ca 4 mm Hg [39]. All the rate constants are much lower than the calculated average dipole orientation (ADO) collision rate constants, which are of the order of 2 × 10 –9 cm3 molecule –1 s –1, consistent with the inhibiting central barrier described by the double-well potential theory [40]. There is excellent agreement between the PHPMS and IMS results for ethyl bromide (EtBr) and i-PrBr, but there is a signiicant difference for methyl bromide (MeBr), which suggests that the difference is not an experimental artifact but might be due to the different pressure regimes. Less eficient stabilization of the initially formed Clˉ···CH3Br complex at the lower pressures could be the explanation. To conirm that the difference is real, Knighton et al. compared the IMS/MS rate constants at ambient pressure, 640 Torr, with PHPMS rate constants at 3 Torr over the temperature range 308–423 K [41]. A signiicantly-higher rate constant was found at the higher pressure, although it was still far below the collision rate. This result, interpreted in terms of the double-well potential theory, suggests that the higher pressure leads to increased stabilization of the short-lived entrance channel intermediate (MeBr)Clˉ*. From the IMS/MS results, the estimated depth of the central barrier below the incoming channel, 2.2 kcal mol–1, is identical with the value obtained from a high level calculation [42].

TABLE 13.3 Rate Constants (cm3 molecule –1 s –1) at 398 K by IMS for the Reaction Cl– + RBr→RCl + Br– Method RBr MeBr EtBr i-PrBr n-BuBr

Area

PHPMSa

MS

3.4 × 10 1.1 × 10–11 8 × 10–13 2.2 × 10–11 –11

3.4 × 10 1.3 × 10–11 7.6 × 10–13 2.2 × 10–11 –11

8.8 × 10–12 9.7 × 10–12 6.2 × 10–13 2.0 × 10–11

Source: Giles, K.; Grimsrud, E.P. J. Phys. Chem. 1992, 96, 6680–6687. a

Caldwell, G.; Magnera, T.F.; Kebarle, P. J. Am. Chem. Soc. 1984, 106, 959–966.

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403

The IMS/MS result cannot conirm that the high-pressure limit of kinetic behavior has been attained, but it demonstrates clearly that low-pressure experiments for such reactions cannot provide deinitive information regarding the problem. There is no difference between the results for the reaction of Clˉ with EtBr and n-BuBr at 640 Torr and at 3 Torr, suggesting that the intermediates in these reactions have longer lifetimes than that of their methyl analog and that their high-pressure limit is attained even at 3 Torr. The upper pressure range of the mobility spectrometer was extended to enable a further study in the reaction of Clˉ with MeBr from 300 Torr to 1100 Torr N2 [43]. Over this range, the reaction rate constant increased by ca 25% demonstrating that the high-pressure limit was not attained even at 1100 Torr. The nascent collision complex must have a lifetime toward back dissociation that is much less than the ca 40 ps between stabilizing collisions. When the nitrogen drift gas was replaced by methane, the rate constant increased further. Better quenching of the intermediate (MeBr)Clˉ* by the more complex drift gas molecule is the most likely explanation for the increase. 13.2.2.3 Type 3. Dissociation of Adduct Ions Rate constants for the irst-order dissociation of symmetrical proton-bound dimers, M2H + → MH +  + M, have been determined for organophosphorus compounds (M = 2,4-dimethylpyridine (DMP) and dimethyl methylphosphonate (DMMP)), where the shapes of the mobility spectra are of the form shown in Figure 13.2d [44]. Some proton-bound dimers decompose in the time taken for the ions to travel between the shutter and the detector plate, and this residence time was varied by changing the electrostatic drift ield strength. Typical ion mobility spectra obtained at different ield strengths are shown in Figure 13.5 and peaks were mass identiied as: irst peak, H + (DMP), the protonated monomer and second peak H + (DMP)2, the proton-bound dimer. The raised baseline between the peaks was due entirely to (DMP)H + , from the decomposition of the proton-bound dimer as in Equation 13.25 H + (DMP)2 → H + (DMP) + DMP

(13.25).

The reaction rate constant was determined in the following manner. The distance x from the shutter at which decomposition occurs at time tx is given by Equation 13.26, in which L is the shutter-detector plate distance, td is the drift time of (DMP)2H +, and tm is the drift time of (DMP)H +   t − tm  x = L  td − tm 

(13.26).

The value of tx is then given by tx =

x xtd  t − tm  = = td   td − tm  vd L

(13.27).

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Intensity (counts)

3.E+05 100 V cm–1 2.E+05 1.E+05 0.E+00 0

5

10

15

20

25

30

35

Drift time (ms)

Intensity (counts)

2.E+06 200 V cm–1

1.E+06

0.E+00 0

5

10

15

20

25

Drift time (ms)

Intensity (counts)

6.E+06 280 V cm–1 3.E+06

0.E+00 0

5

10

15

20

25

Drift time (ms)

FIGURE 13.5 Mobility spectra for DMP at three electric ield strengths obtained in air at 350.4 K and 5 ppm moisture. (Reproduced from Ewing, R.G.; Eiceman, G.A.; Harden, C.S.; Stone, J.A., Int. J. Mass Spectrom. 2006, 255, 76–85. With permission from Elsevier.)

The concentration of ions from proton-bound dimer not decomposed at time tx is proportional to the area of the mobility spectrum from tx to the end of the proton-bound dimer peak. For the irst-order decomposition of M2H + , a plot of the logarithm of this area vs tx is a straight line of slope –k, where k is the reaction rate constant. Examples of plots for the decomposition of (DMP)2H + from measurements obtained at 350 K and different ield strengths are shown in Figure 13.6. The sudden drop in ion signal in each graph signiies the end of the proton-bound dimer peak. The igure demonstrates that the drift time of (DMP)2H + varies inversely with the ield strength, and that the slope is independent of ield strength. Values of k obtained at different temperatures enabled the determination of an activation energy Ea and

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The Study of Ion/Molecule Reactions at Ambient Pressure 18

In [(DMP) 2H* remaining]

17.5

280 260 240 220 200

17 16.5 16

180 160

15.5

140

15 14.5

120 100

14 13.5

0

5

10

15

20

25

tx (ms)

FIGURE 13.6 Plots of ion intensity for (DMP)2H + remaining at time ts at different ield strengths E (V cm–1) at 349 K and 5 ppm moisture in the supporting atmosphere of the drift tube. (Reproduced from Ewing, R.G.; Eiceman, G.A.; Harden, C.S.; Stone, J.A., Int. J. Mass Spectrom. 2006, 255, 76–85. With permission from Elsevier.)

TABLE 13.4 Arrhenius Activation Energies and Pre-exponential Factors for the Reaction M2H + → MH +  + M in the Presence of Different Water Concentrations M DMP DMP DMMP DMMP DMMP

Water (ppmv)

T range (K)

Ea (kJ mol–1)

Log [A (s–1)]

5 2 × 103 5 5 × 102 5 × 103

338–58 311–42 478–98 478–98 478–98

94±2 31±5 127±3 130±2 115±1

15.9±0.4 6.3±0.7 15.6±0.3 15.3±0.4 14.5±0.3

Source: Ewing, R.G.; Eiceman, G.A.; Harden, C.S.; Stone, J.A. Int. J. Mass Spectrom. 2006, 255, 76–85. With permission.

pre-exponential factor A for the dissociation from an Arrhenius plot of ln k vs 1/T. Because some water vapor is always present in mobility drift gases, the experiments presented a favorable opportunity to examine any effects that water vapor may have on reaction rates. The results obtained with different water vapor concentrations are shown in Table 13.4. Of note is the relatively narrow temperature range over which measurements could be made due to the high activation energies for reaction, coupled with the small dynamic range of the mobility spectrometer. The narrow temperature range does not, however, preclude obtaining results with good precision. The pre-exponential factor of ca 1015 s–1 is the maximum expected for unimolecular decompositions [45].

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A measure of the accuracy of the results can be obtained with the knowledge that there should be little or no reverse activation energy for the decomposition, and the activation energy will differ by only RT from the enthalpy of dissociation. The value of Ea obtained for DMP at 5 ppmv water is in excellent agreement with the expected ca 92 kJ mol–1 for symmetrical N2–base dimers [28]. Similarly, the activation energies for DMMP are consistent with the expected ca 134 kJ mol–1 for symmetrical oxygen-base dimers. The activation energy and pre-exponential factor for the decomposition of (DMP)2H + at high water concentration are anomalously low and suggest the inluence of a displacement reaction H 2O + (DMP)2H + → (DMP)H + (H 2O) + DMP

(13.28),

in which the symmetric proton-bound dimer becomes an asymmetric protonbound dimer ((DMP)H + H2O), the activation energy being the difference in bonding enthalpy of DMP and water to (DMP)H + . Lower reduced mobility values for (DMP) H + at the higher water concentration of 2 × 103 ppmv implies, and mass spectra conirmed, that (DMP)H + was indeed hydrated in the experimental temperature range. Data from PHPMS [46] suggest a difference of ca 36 J K–1 mol–1, and the difference of 63 J K–1 mol–1 in Table 13.4 may imply that more than one water molecule is involved in the reaction. The much lower entropy change expected for a displacement reaction compared with unimolecular dissociation is also consistent with the lower pre-exponential factor. The dissociation of (DMMP)2H + occurred at a much higher temperature than that of (DMP)2H + , and the slight effect of water vapor on the kinetics shows that the reaction was mainly unimolecular even at 5 × 103 ppmv water.

13.2.3

THE KINETICS OF THERMAL ELECTRON CAPTURE AND THERMAL ELECTRON DETACHMENT

Ion sources operating at atmospheric pressure in N2 or air provide an abundant source of thermalized electrons. In the absence of molecules with suitable electron afinity, and with an ion mobility spectrometer operating in the negative ion mode, electrons are injected into the drift region to arrive at the detector about 100 times faster than any ion. Suitable molecules present in the drift region may capture electrons, leading to a decrease in electron signal and a negative ion signal at much longer times. Anions formed in the source region by attachment of thermalized electrons, and injected into the drift region in the absence of attaching molecules, may thermally lose the electron at suitable temperatures and produce an interpretable mobility spectrum. Mobility spectrometers operating at atmospheric pressure have been employed to obtain reaction rate constants for both electron capture and for thermal electron detachment studies and these methods are discussed separately below. 13.2.3.1 Electron Capture Spangler and Lawless [47] measured the rate constant for dissociative electron capture by chlorobenzene by monitoring the production of Clˉ. Electrons traveling down the drift tube to the detector encountered chlorobenzene molecules from an exponential dilution

The Study of Ion/Molecule Reactions at Ambient Pressure

407

lask whose concentration varied in a well-deined manner. The Clˉ ion signal was related to the chlorobenzene concentration and a rather complex mathematical modeling of the system gave a rate constant of 7.1 (±3.1) × 10 –11 cm3 s–1 at 473 K, in agreement with experimental data from the more conventional swarm beam technique [48]. Mayhew and co-workers constructed a mobility spectrometer–mass spectrometer combination that has been used extensively to determine the kinetics of the attachment of low energy electrons to halogen-containing molecules at atmospheric pressure [49–56]. Attachment rate coeficients have been determined in two ways. One method is by monitoring the attenuation of a pulse of electrons passing through the drift region containing a known concentration of attaching molecules, and the second by determining the axial distribution of the resulting anions. Excellent agreement between the results obtained by the two methods and with results from the literature was found for SF6 [56]. A pulse of electrons passing through the drift region is attenuated exponentially by radial scattering to give a detector signal Io. In the presence of a constant concentration of an attaching molecule M the signal is further attenuated to a value I, as in I = I oe − αL [ M ]

(13.29),

where α is the density-normalized electron attachment coeficient and L is the drift length. A plot of ln(I/Io) vs the concentration of M has slope –αL. The rate constant for electron attachment k is obtained from αL with the substitution L = wt where t is the electron arrival time and w is the mean electron drift velocity. The value of w, which is a function of E/N, is different for each drift gas and is determined theoretically (values for nitrogen are available in the literature [37]). The electron attachment rate constant for SF6 in nitrogen at ambient temperature and pressure showed a smooth decline with increasing E/N over the range of 0.39– 0.78 Td [56]. As shown in Figure 13.7, the results obtained by IMS agree closely with those obtained by the well-established high-pressure swarm technique [57]. A further series of experiments with E/N from 0.05 to 0.9 Td conirmed this excellent agreement between the two methods [55]. Both the mobility and the swarm experiments showed that the electron energy distribution in nitrogen is not thermal, even at E/N  M Th are ejected resonantly with a broadband waveform. Figure 15.17 shows the scan function of this isolation method. In this method, ions with mass/charge ratio ... 802.0>500:8... 880.0>600... 960.0>700:970 [2...

10

100

5–1 4

KCounts

75

5–2 6–1

3

7

6–2

50

8

25

9

0 7

8

9

10 Minutes

11

12

13

FIGURE 15.44 PBDE MS/MS Chromatogram, 50 ppb concentration (100 ppb for BDE 209). See Table 15.2 for peak names.

TABLE 15.2 The Analysis of PBDE Congeners in Accordance with the RoHS Regulation. The Peak Number in the Table Refers to the Peaks Labeled in Figure 15.44 Peak Number 3 4 5–1 5–2 6–1 6–2 7 8 9 10

PBDE Isomer BDE 28 BDE 47 BDE 99 BDE 100 BDE 153 BDE 154 BDE 183 BDE 205 BDE 206 BDE 209

15.3.3.4

Ion Trap Analysis with Liquid Chemical Ionization (CI) Reagents: USEPA Method 521 Ion traps can be conigured readily to use liquid reagents for CI directly inside the ion trap cavity. Low vapor pressure liquids, such as acetonitrile (CH3CN) or methanol (CH3OH), can be used as CI reagents. The sequence of reactions is as follows: CH3CN + e – → CH3CN+• + 2e –

(15.2)

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(a)

(b) RoHS Matrix 4, taken between 10 other matrix injections RSD 10.8% Raw Area

Peak Area

150000 100000

Series1

50000 0

1

2

3

4 5 6 7 Injection Number

8

9

10

FIGURE 15.45 Decabromodiphenyl ether, congener BDE-209: (a) upper, total ion chromatograph showing detection of BDE-209 in an acrylonitrile butadiene styrene (ABS) plastic extract; (a) lower, product ion mass spectrum of BDE-209 showing the loss of Br2 from each of the ions of the molecular ion cluster; (b) %RSD in the chart is obtained from raw peak area data for a standard solution run after 10 injections of the ABS extract.

CH3CN+• + CH3CN → CH3CNH+ + CH2CN•

(15.3)

CH3OH + e – → CH3OH2+• + 2e –

(15.4)

CH3OH +• + CH3OH → CH3OH2+ + CH3O•

(15.5)

where the protonated molecules CH3CNH + and CH3OH2+ are CI reagent ions. The basic steps are: (a) form and trap reagent ions in the ion trap; (b) remove unwanted ions formed by EI using applied waveforms, leaving only CI reagent ions

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TABLE 15.3 The Accuracy and Precision of the Seven Nitrosamines Listed in EPA Method 521 Using the CI/MS/MS Technique Compound Name NDMA NMEA NDEA NDPA NPYR NPIP NDBA

Meas.Conc. (ppb)

Accuracy (%)

0.934 0.986 0.997 1.056 0.904 0.924 0.896

93.4 98.6 99.7 105.6 90.4 92.4 89.6

Precision (RSD%) 7.30 11.49 3.08 10.09 5.08 4.55 5.42

Note: These data are based upon 11 injections of a standard solution of each Nitrosamine at a concentration of 1 ppb.

in the ion trap; (c) reagent ions react with sample to form protonated molecules or adduct ions; and (d) ions ejected in sequential mass order to form the mass spectrum. The technique is applicable particularly to compounds that yield multiple fragments (thus low response) under normal EI conditions. It is also useful for identifying and/ or conirming the molecular weight of a compound. USEPA Method 521 is used to determine the concentration of certain nitrosamines in source water and inished drinking water. Early health effects or toxicity data suggest that nitrosamines, in general, are powerful carcinogens [62]. Therefore, there is a need for an analytical method for the detection of nitrosamines at very low concentrations (parts-per-trillion levels or ppt) in inished drinking waters and source waters. Nitrosamines under normal EI conditions have very poor response, with multiple fragment ions being formed of low mass/charge ratio. CI provides protonated ions with much better response, and MS/MS adds speciicity to the analysis. Nitrosamines provide a good example of how a speciic ion trap technique can solve a tough analytical problem. These compounds are extracted from the water sample using a form of activated carbon, so matrix interference can become signiicant in waters that contain a high total organic carbon (TOC) content. The data shown in Table 15.3 illustrate the excellent accuracy and precision of the CI/MS/MS technique for the seven nitrosamines at a concentration of 1 ppb. These data are based upon 11 injections of a standard solution. A TIC of typical target compounds, to which the CI/MS/MS technique is applied, is shown in Figure 15.46. 15.3.3.5

Analysis of Polychlorinated Biphenyls (PCBs) by Ion Trap Mass Spectrometry Polychlorinated biphenyls (PCBs) exist as 209 individual congeners, exhibiting all the variations of position and number of chlorine substitutions in a biphenyl molecule.

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250

NPYR

NMEA

NDBA

NDEA

200

kCounts

NPIP NMOR

150 100 50

NDMA NDPA

NDMA-d6(Surr.)

NDPA-d14((IS)

0 15

20

Minutes

25

30

FIGURE 15.46 CI/MS/MS TIC of some common nitrosamines listed in USEPA 521. Extract concentration is 50 ppb for each nitrosamine.

Each PCB congener is named according to the positions of chlorine substitution on the two phenyl rings of biphenyl, and the toxicity of PCBs correlates strongly with their structures. The toxicity of PCB mixtures is due principally to a small group of non-ortho and mono-ortho-substituted congeners [63]. USEPA Method 505 [64] is used for the determination of PCBs and other pesticides in both ground and surface water. A sample is prepared by extracting a small volume, typically 40 mL, with 2 mL of hexane. The hexane layer is removed and analyzed by gas chromatography. The method uses electron capture detection (ECD) for added sensitivity for halogenated compounds. Although ECD is a very sensitive detector for this analysis, it is prone to matrix interference and can result in false positive identiication due to co-eluting peaks. Mass spectrometry can overcome this problem, however the sensitivity of the technique in full mass scan or SIM can be challenging in relation to the micro-extraction sample preparation procedure described in EPA Method 505. Current ion trap mass spectrometers, using a full mass scan for many compounds, can obtain very similar sensitivity to that achievable with ECD. Figure 15.47 illustrates the detection and identiication, by ion trap mass spectrometry, of PCB isomer 2,3-dichlorobiphenyl at a concentration of 0.05 ppb (1.75 pg injected on-column). The PCBs were extracted using the micro-extraction technique listed in USEPA Method 505. Tables 15.4 and 15.5 illustrate that the compounds can be detected at levels similar to those attainable with ECD with excellent precision and accuracy. A set of replicates, spiked at low concentrations in the aqueous sample (0.025 to 0.25 ppb) of polychlorinated biphenyl (PCB) congeners in reagent and surface

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FIGURE 15.47 2,3-Dichlorobiphenyl at a concentration of 0.05 ppb in the aqueous sample (0.75 pg injected on-column). The upper half of the igure is an extracted ion mass chromatogram for m/z 152 + m/z 222. The lower half of the igure is a full scan mass spectrum of the same peak.

TABLE 15.4 Calibration Ranges Studied by Ion Trap Mass Spectrometry PCBs mono, di and tri tetra, penta and hexa hepta and octa deca

Aqueous Concentration (ppb)

Conc. on Column (pg)

0.025–5 0.05–10 0.075–15 0.125–25

0.875–175 1.75–350 2.625–525 4.375–875

Note: The aqueous concentration refers to the inal concentration of the listed PCB isomers spiked into laboratory reagent water and extracted using the procedure described in EPA method 505. The second column lists the actual amount of the PCB isomers that were injected into the analytical system from the resulting aqueous extractions.

waters, was extracted and run in EI full mass scan mode for the determination of Method Detection Limits (MDL). The MDL in USEPA Method 505 is calculated based on the standard deviation of quantitative results multiplied by Student’s t at 99% conidence level [65].

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

TABLE 15.5 Method Detection Limits (MDLs) of PCBs by Full Scan Ion Trap Mass Spectrometry in Reagent Water and Surface Water PCBs

Reagent Water (n = 8)

Surface Water (n = 16)

mono di tri tetra penta hexa hepta octa deca

0.010 0.011 0.010 0.009 0.017 0.016 0.014 0.017 0.082

0.010 0.015 0.014 0.024 0.053 0.040 0.068 0.085 0.221

Note: The units are µg L–1. A total of eight spiked samples for reagent water and 16 spiked samples for surface water were used in the statistical calculations based upon the MDL procedure outlined in EPA Method 505.

15.4

SUMMARY

Since the middle 1990s, numerous efforts on fully-AMDs have made GC/MS and GC/MS/MS ion trap mass spectrometers to be practical and mature commercial products. Since then, GC/MS and GC/MS/MS ion traps have grown from novel research instruments into routine, widely-applied analytical instruments. GC/MS and GC/MS/MS ion trap instruments have reached a high level of maturity as they have grown in the directions of higher performance, such as faster scan speed, higher mass resolution, and extended charge capacity. The advent of the high-performance linear ion trap has provided the opportunity for GC/MS and GC/MS/MS to grow even further in the future. The GC/MS applications discussed above demonstrate clearly that ion traps provide excellent data for applications, despite a history of poor performance in early ion trap designs. Qualitative and quantitative analysis in heavy matrices are possible because increased ion trapping capacity and ion population control is available in modern instrumentation. The technology has been accepted for use with major USEPA methods as a routine analytical tool for challenging environmental samples. The analyzer is versatile, because scan modes such as MS/MS, liquid CI, hybrid CI, and full scan mass spectrometry can be performed on the same instrument. This instrumental versatility reduces cost and increases speciicity by providing more information about the molecules under study.

ACKNOWLEDGMENTS The authors would like to thank Dr. Barbara Bolton, Dr. Kenneth Newton, and Dr. Haibo Wang for their helpful discussions and useful data.

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REFERENCES 1. Paul, W.; Steinwedel, H. Apparatus for Separating Charged Particles of Different Speciic Charges, US Patent 1960, 2,939,952. 2. March, R.E.; Todd, J.F.J. Quadrupole Ion Trap Mass Spectrometry, Second edition of Quadrupole Storage Mass Spectrometry. Chemical Analysis Series, vol. 165. John Wiley, Hoboken, New Jersey, 2005. 3. Stafford, G.C., Jr.; Kelley, P.E.; Stephens, D.R. Method of Mass Analyzing a Sample by Use of a Quadrupole Ion Trap, US Patent 1985, 4,540,884. 4. Stafford, G.C., Jr.; Kelley, P.E.; Syka, J.E.P.; Reynolds, W.E.; Todd, J.F.J. Recent improvements in and analytical applications of advanced ion trap technology. Int. J. Mass Spectrom. Ion Processes. 1984, 60, 85–98. 5. Kelley, P.E.; Stafford, G.C. Jr.; Syka, J.E.P.; Reynolds, W.E.; Louris, J.N., Amy, J.W.; Todd, J.F.J. New advances in the operation of the ion trap mass spectrometer. Proc. 33rd ASMS Conference on Mass Spectrometry and Allied Topics, San Diego, CA, 1985, 707–708. 6. Syka, J.E.P.; Louris, J.N.; Kelley, P.E.; Stafford, G.C. Jr.; Reynolds, W.E. Method of Operating Ion Trap Detector in MS/MS Mode, US Patent 1988, 4,736,101. 7. Louris, J.N.; Schwartz, J.C.; Stafford, G.C. Jr.; Syka, J.E.P.; Taylor, D.M. The Paul ion trap mass selective instability scan: trap geometry and resolution. Proc. 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington DC, 1992, 1003–1004. 8. Franzen, J. Simulation study of an ion cage with superimposed multiple ields. Int. J. Mass Spectrom. Ion Processes. 1991, 106, 63–78. 9. Splendore, M.; Marquette, E.; Oppenheimer, J.; Huston, C.; Wells, G.J. A new ion ejection method employing an asymmetric trapping ield to improve the mass scanning performance of an electrodynamic ion trap. Int. J. Mass Spectrom. 1999, 190/191, 129–143. 10. Wells, G.J.; Wang, M.; Marquette, E.G. Mass Scanning Method Using an Ion Trap Mass Spectrometer, US Patent 1998, 5,714,755. 11. Schwartz, J.C.; Senko, M.W.; Syka, J.E.P. A two-dimensional quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 2002, 13, 659–669. 12. Stafford, G.C. Jr.; Taylor, D.M. Method of Increasing the Dynamic Range and Sensitivity of a Quadrupole Ion Trap Mass Spectrometer, US Patent 1992, 5,107,109. 13. Schwartz, J.C. Do space charge effects limit LC quadrupole ion trap performance. Proc. 9th Sanibel Conference on Mass Spectrometry, Sanibel Island, FL, 1997. 14. Brekenfeld, A.; Kaplan, D.A.; Hartmer, R.; Wilson, J.; Gebhardt, C.; Schubert, M. The right place and the right time: increasing the capacity in modern 3-D ion traps. Proc. 55th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, IN, 2007. 15. Mordehai, A.; Miller, B.; Bai, J.; Brekenfeld, A.; Baessmann, C.; Schubert, M.; Hosea, K. Improved 3D ion trap-ion detector coupling and techniques for evaluating exact ion trap capacity. Proc. 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 2004. 16. George, J.E.; III, Specht, A.; Newton, K.; Wells, G. A comparison of the spectral charge capacity between two ejection methods. Proc. 54th ASMS Conference on Mass Spectrometry and Allied Topics, Seattle, WA, 2006. 17. Libong, D.; Bruneau, S.P.C.; Rogalewicz, F.; Ricordel, I.; Bouchonnet, S.J. Adsorptiondesorption effects in ion trap mass spectrometry using in situ ionization. Chromatog. 2003, 1010 (issue 1), 123–128. 18. Taylor, D.M.; Amy, J.W.; Stafford, G.C. Jr. Metal Surfaces for Sample Analyzing and Ionizing Apparatus, US Patent 1991, 5,055,678.

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19. Brittain, R.; Wang, M. Surface Coating to Improve Performance of Ion Trap Mass Spectrometers, US Patent 1997, 5,633,497. 20. McLuckey, S.A.; Glish, G.L.; Asano, K.G.; Van Berkel, G.L. Self chemical ionization in an ion trap mass spectrometer. Anal. Chem. 1988, 60, 2312–2314. 21. Booth, M.M.; Stephenson, J.L.J.; Yost, R.A. Gas chromatography/ion trap mass spectrometry using an external ion source. Proc. 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, 1993, 716a–716b. 22. Bier, M.E.; Syka, J.E.P.; Taylor, D.M.; Fies, W.J. Ion Source Assembly for an Ion Trap Mass Spectrometer and Method, US patent 1998, 5,756,996. 23. Wells, G J.; Yee, P.P.; Ruport, M.A.; Huston, C.K. Pulsed Ion Source for Ion Trap Mass Spectrometer, US Patent 2001, 6,294,780. 24. Louris, J.N.; Syka, J.E.P.; Kelley, P.E. Method of Operating Quadrupole Ion Trap Chemical Ionization Mass Spectrometry, US Patent 1987, 4,686,367. 25. Strife, R.J.; Keller, P.J. Ion trap ionization mass spectrometry-RF/DC for isolating unique reactant ions. Org. Mass Spectrom. 1989, 24(3), 201–204. 26. March, R.E.; Todd, J.F.J. eds. Practical Aspects of Ion Trap Mass Spectrometry. Vol. 3, “Chemical, Environmental, and Biomedical Application”, Ch. 7, “Chemical Ionization in Ion Trap Mass Spectrometry”, p. 239–253, by Creaser, C.S. Modern Mass Spectrometry series, CRC Press, Roca Baton, FL, 1995. 27. Berberich, D.W.; Heil, M.V.; Johnson, J.V.; Yost, R.A. Mass-selection of reactant ions for chemical ionization in quadrupole ion traps and triple quadrupole mass spectrometers. Int. J. Mass Spectrom. Ion Processes. 1989, 94, 115–147. 28. Van Pelt, C.K.; Carpenter, B.K.; Brenna, J.T. Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters. J. Am. Soc. Mass Spectrom. 1999, 10, 1253–1262. 29. Brandt, S.D.; Freeman, S.; Fleet, I.A.; Alder, J.F. Analytical chemistry of synthetic routes to psychoactive tryptamines Part III. Characterisation of the Speeter and Anthony route to N,N-dialkylated tryptamines using CI-IT-MS-MS. Analyst 2005, 130, 1258–1262. 30. Hunt, D.F.; Stafford, G.C.; Crow, F.W.; Russell, J.W. Pulsed positive negative ion chemical ionization mass spectrometry. Anal. Chem. 1976, 48, 2098–2104. 31. March, R.E.; Todd, J.F.J. eds. Practical Aspects of Ion Trap Mass Spectrometry. Vol. 3, Ch. 2, “Ion Trap as Tandem Mass Spectrometers”, pp. 27–88, by March, R.E.; Strife, R.J.; Creaser, C.S. Modern Mass Spectrometry series, CRC Press, Roca Baton, FL, 1995. 32. March, R.E.; Todd, J.F.J. eds. Practical Aspects of Ion Trap Mass Spectrometry. Vol. 3, Ch. 4, “Practical Ion Trap Technology: GC/MS and GC/MS/MS”, pp. 121–185, by Yates, N.A.; Booth, M.N.; Stephenson, J.L., Jr.; Yost, R.A. Modern Mass Spectrometry series, CRC Press, Roca Baton, FL, 1995. 33. Wells, G.J. Quadrupole Trap Improved Technique for Ion Isolation, US Patent 1993, 5,198,665. 34. Marshall, A.G.; Ricca, T.L.; Wang, T.L. Tailored Excitation for Trapped Ion Mass Spectrometry, US Patent 1988, 4,761,545. 35. Louris, J.N.; Taylor, D.M. Method and Apparatus for Ejecting Unwanted Ions in an Ion Trap Mass Spectrometer, US Patent 1994, 5,324,939. 36. Kelley, P.E. Mass Spectrometry Method Using Notch Filter, US Patent 1992, 5,134,286. 37. Buttrill, S.E. Jr. Quadrupole Ion Trap Method Having Improved Sensitivity, US Patent 1994, 5,300,772. 38. Wang, M.; Lee, D.; Newton, K.; Schachterle, S. High-Resolution Ion Isolation Utilizing Broadband Waveform Signals, US Patent 2008, 7,378,648. 39. Schwartz, J.C.; Syka, J.E.P.; Quarmby, S.T. Improving the fundamentals of MSn on 2D linear ion traps: new ion activation and isolation techniques. Proc. 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, 2005.

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40. Salmon, K.; He, M.; Choudhary, G.; Schwartz, J.; Cho, D. Improved isolation eficiency using higher resolution isolation in an ion trap mass spectrometer. Proc. 54th ASMS Conference on Mass Spectrometry and Allied Topics, Seattle, WA, 2006. 41. Wells, G.J. Quadrupole Trap Improved Technique for Collisional Induced Disassociation for MS/MS Process, US Patent 1994, 5,302,826. 42. Jackson, G.P.; Hyland, J.J.; Laskay, U.A. Energetics and eficiences of collision-induced dissociation achieved during the mass acquisition scan in a quadrupole ion trap. Rapid Commun. Mass Spectrom. 2005, 19, 3555–3563. 43. Schwartz, J.C.; Taylor, D.M. Method of Ion Fragmentation in a Quadrupole Ion Trap, US Patent 2000, 6,124,591. 44. Mulholland, J.J.; Yost, R.A. Multi-level CID: a novel approach for improving MS/MS on the quadrupole ion trap. Proc. 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, 1999. 45. Salmon, K.; Choudhary, G.; Schwartz, J.; Cho, D. Enhanced fragmentation of small molecules in a linear ion trap mass spectrometer using stepped normalized collision energy. Proc. 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, 2005. 46. Brekenfeld, A.; Schubert, M.; Franzen, J. Fragmentation in Quadrupole Ion Trap Mass Spectrometers, US Patent 2002, 6,410,913. 47. Goodley, P.C. Technical Note, 5988-0704EN, 2000, Agilent Technologies. 48. Wang, M. Chemical Structure Insensitive Method and Apparatus for Dissociating Ions, US patent application, publication pending. 49. Cunningham, C., Jr.; Glish, G.L.; Burinsky, D.J. High amplitude short time excitation: a method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 2006, 17, 81–84. 50. Schwartz, J.C. High-Q Pulsed Fragmentation in Ion Trap, US Patent 2005, 6,949,743. 51. SW-846, Method 8270D, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, available from: National Technical Information Services, US Department of Commerce, 5285 Port Royal Road, Springield, VA 22161. 52. SDWA: Safe Drinking Water Act, EPA 816-F-04-030, June 2004, “Understanding the Safe Drinking Water Act”. 53. 525.2 Methods for the Determination of Organic Compounds in Drinking WaterSupplement III (EPA/600/R-95-131). Citation Information Methods for the Determination of Organic Compounds in Drinking Water-Supplement III (EPA/600/R-95-131). This document is available through NTIS (http://www.ntis.gov). Alternatively, the methods from this source can be found on the following CD-ROM: EPA Methods and Guidance for Analysis of Water, Version 2.0. 54. EPA Method Guidance CD-ROM (includes MCAWW Methods, and most current EPA Methods) Citation Information EPA Methods and Guidance for Analysis of Water, Version 2.0 ’ This CD-ROM includes all EPA wastewater test methods approved at 40 CFR 136, all EPA drinking water test methods approved at 40 CFR 141, and various EPA guidance documents related to EPA’s wastewater and drinking water programs. New and revised EPA OW methods and guidance documents will be added to the CD-ROM during periodic updates. Web at: http://www.ntis.gov/product/environmental-test-methods.htm 55. Price, E.K.; Prakash, B.; Domino, M.M.; Pepich, B.V.; Munch, D.J. 2005, Determination of selected pesticides and lame retardants in drinking water by solid phase extraction and capillary column gas chromatography/mass spectrometry: U.S. Environmental Protection Agency Report EPA/815/R-05/005, Version 1.0. 56. Methods for the Determination of Organic and Inorganic Compounds in Drinking Water, Volume 1 (EPA/815-R-00-014) Citation Information Available through: NSCEP Item # 815-R-00-014, (800) 490-9198 or (513) 489-8190 or order from the Web at: http://www. epa.gov/ncepihom/

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57. Munch, J.W. Revision 1.0, Sept. 2002, NERL Method 529: Determination of Explosives and Related Compounds in Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography/Mass Spectrometry (GC/MS). 58. Munch, J.W. Revision 1.0, Sept. 2004, NERL Method 521: Determination of Nitrosamines in Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography with Large Volume Injection and Chemical Ionization Tandem Mass Spectrometry (MS/MS). 59. SW-846, Method 8270D, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, available from: National Technical Information Services, US Department of Commerce, 5285 Port Royal Road, Springield, VA 22161 60. Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and dispersive solid phase extraction for the determination of pesticide residues in produce. (QuEChERS method). J. AOAC Int. 2003, 86, 412–431. 61. The Directive on the Restriction of the Use of Certain Hazardous Substances in Electrical and Electronic Equipment 2002/95/EC[1] http://eur-lex.europa.eu/LexUriServ/Lex UriServ.do?uri = OJ:L:2003:037:0019:0023:EN:PDF 62. Lijinsky, W.; Epstein, S.S. Nitrosamines as Environmental Carcinogens. Eppley Institute for Research in Cancer, University of Nebraska College of Medicine, www.nature.com/ nature/journal/v225/n5227/abs/225021a0.html 63. Tanabe, S. PCB Problems in the Future: Foresight from Current Knowledge. Environ. Pollut. 1988. 50, 5–28. 64. Method 505: Analysis of Organohalide Pesticides and Commercial Polychlorinated Biphenyls (PCB) Products in Water by Microextraction and Gas Chromatography, Winield, T.W.; Munch, J.W. 1995. National Environmental Research Laboratories, Ofice of Research and Development, USEPA, Cincinnati, OH 45268. 65. Method 505: Analysis of Organohalide Pesticides and Commercial Polychlorinated Biphenyls (PCB) Products in Water by Microextraction and Gas Chromatography, Winield, T.W.; Munch, J.W. 1995, pp.18–19. Note: EPA Method 505 from: Methods for the Determination of Organic Compounds in Drinking Water-Supplement III (EPA/600/R-95-131). This document is available through NTIS (http://www.ntis.gov). Alternatively, the methods from this source can be found on the following CD-ROM: EPA Methods and Guidance for Analysis of Water, Version 2.0.

Monitoring 16 Remote of Volatile Organic Compounds in Water by Membrane Inlet Mass Spectrometry Romina Pozzi, Paola Bocchini, Francesca Pinelli, and Guido C. Galletti CONTENTS 16.1 16.2 16.3

Introduction ................................................................................................ 492 Membrane Inlet Mass Spectrometry (MIMS) ............................................ 493 Membrane Inlet Mass Spectrometry (MIMS) Instrumentation for Prolonged Monitoring ................................................................................. 494 16.3.1 Experimental ................................................................................ 494 16.3.2 Laboratory Tests ........................................................................... 496 16.3.3 Field Tests ..................................................................................... 496 16.4 Results and Discussion ............................................................................... 497 16.4.1 Laboratory Tests ........................................................................... 497 16.4.1.1 Detection Limits (LOD)............................................... 497 16.4.1.2 Reproducibility ............................................................ 498 16.4.1.3 Linearity....................................................................... 498 16.4.1.4 Matrix Effects .............................................................. 499 16.4.2 Case Studies .................................................................................. 499 16.4.2.1 Acrylonitrile ................................................................. 499 16.4.2.2 Comparison of MIMS with Purge-and-Trap (P&T)/Gas Chromatography (GC)/ Mass Spectrometry (MS) .............................................500 16.4.3 Field Tests ..................................................................................... 502 16.5 Conclusion .................................................................................................. 505 References ..............................................................................................................506

491

492

16.1

Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

INTRODUCTION

European Union Directive 98/83 underlines the importance of determining the quality of drinking water in order to protect human health. In particular, a number of chemical compounds are listed, such as benzene, 1,2-dichloroethane, tetrachloroethylene, chloroform, and trihalomethanes, whose concentration in drinking water must be kept under well-deined thresholds. In Italy, laws DL 31/01, DM 152/99, and DM 471/99 set the norms for the concentrations of these, and of many other compounds in drinking water, wastewater, and contaminated sites, respectively. Volatile Organic Compounds (VOCs) constitute a very important class of water pollutants because of their persistence; in addition, many of them are suspected of being carcinogenic. There are about 60 VOCs, including benzene, toluene, ethylbenzene, and xylenes (‘BTEX compounds’), halomethanes, and haloethanes. The presence of some of them in water is due to anthropic activities, for example, the use of chlorinated solvents in industries and laundries, and the formation of halomethanes as by-products of water disinfectants. With respect to Italian law DL 31/01, the maximum allowable concentration (threshold) for the sum of trichloroethylene and tetrachloroethylene concentrations in drinking water is 10 ppb, whereas the minimum account for the sum of a set of four halogenated compounds, namely chloroform, bromoform, bromodichloromethane, and chlorodibromomethane must be as low as possible and must not exceed 30 ppb. Note that 30 ppb is equivalent to 30 µg L –1. A real-time, on-line, continuous monitoring system for such compounds would allow either prompt actions to be taken in order to avoid the diffusion of pollutants into the water system or to take appropriate countermeasures, thus restoring safe conditions in the case of accidental contamination. In general, only the conventional chemical-physical parameters, such as dissolved oxygen temperature, pH, conductivity, and turbidity, are monitored continuously in water [1]. VOCs are usually analysed in the laboratory by means of Purge and Trap/Gas Chromatography/ Mass Spectrometry (P&T/GC/MS) using the U.S. Environmental Protection Agency (USEPA) Method No. 8260B which sets the standard for the analysis of VOCs in water. Although the method is state-of-the-art in terms of sensitivity, reproducibility, validation of the overall procedure and has been adopted worldwide by water laboratories, it can by no means be considered an alarm tool giving rapid warning of concentration increases. For an analytical procedure to be considered a warning device, it should be rapid, simple, and able to work unattended 24-hours-a-day for several days in unmanned sites and to send remotely analytical reports. As appropriately stated by Mikkelsen and coworkers, reporting upon a robust and sensitive on-line remote monitoring system for heavy metals in natural waters, “it is a great distance from developing a method (…) for continuous outdoor measurements” [2]. To our knowledge, little research has been made on the use of the ion trap for continuous, on-line monitoring of environmental parameters. Masuyoshi Yamada et al. studied a continuous monitoring system for the determination of polychlorinated biphenyls in air; the system employed direct sampling atmospheric pressure chemical ionization (APCI)/ion trap mass spectrometry (ITMS) [3]. Direct sampling ion trap mass spectrometers with two direct sampling interfaces, developed at Oak Ridge National Laboratory, TN, USA,

Remote Monitoring of Volatile Organic Compounds in Water

493

have been tested in ield studies to determine VOCs in the efluents from hazardouswaste incinerators [4]. Kurten et al. developed an ion trap mass spectrometer for the on-line chemical analysis of atmospheric aerosol particles [5].

16.2 MEMBRANE INLET MASS SPECTROMETRY (MIMS) Riter et al. applied Membrane Inlet Mass Spectrometry (MIMS) coupled to a miniature mass spectrometer equipped with a cylindrical ion trap (CIT) analyzer to monitor the lavor components directly from human breath [6]. Johnson et al. measured ethanol concentrations on-line in fermentation broths from a 9000-L fermentation reactor for a period of four days [7]. However, data reported in the above papers referred to experiments lasting no more than a few days. MIMS has been extensively studied for the determination of VOCs in various environmental matrices, especially water and air samples [8–16]. Ketola and coworkers published a review that listed 172 references of MIMS applications to water and air [17]. MIMS allows the introduction of VOCs to the mass spectrometer through a thin (some tenths of a millimeter) hollow-iber polymeric membrane, which is selective toward organic compounds. When the membrane is in contact with the sample and an ion trap mass spectrometer is used as the detector, such as in the case here, VOCs are extracted into the membrane, concentrated in its small volume, and swept into the mass spectrometer by a gentle stream of helium carrier gas. The whole process is called pervaporation and is divided into three steps: (a) phase-partitioning equilibrium of the organic compound between the sample (water or air) and the membrane; (b) diffusion of the compound by a concentration gradient from the outer side of the membrane (in contact with the sample) to the inner side (connected to the mass spectrometer); and (c) evaporation of the compound from the inner side of the membrane [18]. Diffusion is the rate-determining step, whereas partitioning and evaporation can be considered to be instantaneous. When the membrane is exposed to a sample containing the target compounds and the ions characteristic of each target compound are detected by mass spectrometry, the inherent ion current increases up to a plateau showing that the analyte’s pervaporation rate and transport low to the detector are equal. The pervaporation process can be described by the Fick’s equations of diffusion [18], that is,  ∂ Cm ( x , t )  I m ( x , t ) = − AD    ∂ x 

(16.1),

 ∂ Cm ( x , t )   ∂ 2C m ( x , t )   = D     ∂ t  ∂ x2 

(16.2)

where: Im = analyte low through the membrane (mol s–1); Cm = concentration of the analyte in the membrane wall (mol cm–3); A = membrane surface (cm2); D = diffusion coeficient (cm2 s–1);

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

x = membrane thickness (cm); t = time (s). The characteristic ions are the qualitative information which allows identiication of the analytes, while plateau height (Iss = ADCm /L, where Iss is the analyte low and L is the membrane thickness) is the quantitative information, with sensitivity in the sub-ppb levels and dynamic range of up to four decades for many VOCs [17,19].

16.3

MEMBRANE INLET MASS SPECTROMETRY (MIMS) INSTRUMENTATION FOR PROLONGED MONITORING

Although the many papers cited above have demonstrated that MIMS is a potentially excellent technique for continuous VOC monitoring given its simplicity and sensitivity, to our knowledge no account has been published of experimental attempts to demonstrate that MIMS can really be implemented in a device able to work unattended for months. Following our previous paper on MIMS upgrades [19], this present work reports on laboratory and ield tests of hardware and software for MIMS instruments built in our laboratory. Four instruments were deployed in unmanned sites, where they monitored VOCs in natural waters and wastewater during a period exceeding one year for each instrument. The instruments were equipped with software that facilitated the automatic operation of each analysis, the identiication and quantitation of VOCs from the raw mass spectra, and the transmission of the results to a remote control room via internet connection. In the remote control room, a personal computer with dedicated software displayed the results as bar graphs and was programed to activate alarms when set concentration thresholds were exceeded. Laboratory performance in terms of sensitivity, reproducibility, linearity tests, and comparison with P&T/GC/MS together with ield performance in terms of data output, most frequent maintenance operations and technical failures, and overall stability of the four remotely-controlled instruments are discussed.

16.3.1

EXPERIMENTAL

Table 16.1 lists the VOCs used in the present study together with their respective characteristic ions. All compounds were purchased from Sigma-Aldrich (St Louis, MO, USA). The MIMS system (Analytical Research Systems, Bologna, Italy) was equipped with a helium carrier gas cylinder (chromatography grade, SIAD, Milan, Italy), pressure regulator and a 30 m column with no stationary phase to provide a constant gas low (1 mL min–1), sample cell, and hollow iber membrane connected to a quadrupole ion trap mass spectrometer (Varian Inc., Walnut Creek, USA) through a fused silica column (0.32 mm ID, 5 m, Supelco) without a stationary phase. All mass spectra were acquired (5 min) from m/z 50 to 200 at a rate of 1 spectrum/5 s. The trap temperature was 170°C. The sample was kept under magnetic stirring at room temperature during the analysis. Each instrument had ive sample inlets so that up to ive different water streams could be analyzed. During normal operating conditions, two inlets were dedicated to blank water and calibration solutions, respectively. The device was operated by means of proprietary software able to set: (a) sampling, analytical, and data-transmission functions; (b) identiication and

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Benzene Toluene Ethylbenzene Cumene Styrene 1,4-dichlorobenzene 1,2-dichlorobenzene Chloroform Trichloroethylene Tetrachloroethylene Carbon tetrachloride Bromoform Dibromochloromethane Dichlorobromomethane 1,1,1-Trichloroethane Acrylonitrile

Compound

78 91 91 + 106 77 + 105 + 120 78 + 104 146 + 148 + 150 146 + 148 + 150 83 130 + 132 164 + 166 117 + 119 173 129 83 96 + 97 52

Characteristic Ions (m/z) 0.05 0.3 0.1 9 0.2 0.2 0.2 0.03 0.03 0.08 0.1 0.20 0.1 0.1 0.09 40

LOD

MIMS

7 9 9 9 6 5 5 6 4 8 16 19 9 6 10 11

SD% 0.9996 0.9998 0.9998 0.9087 0.9922 0.9986 0.9986 0.9977 0.9984 0.9994 0.9965 0.9927 0.9986 0.9964 0.9982 1.0000

R2 0.25 0.25 0.1 0.25 0.5 0.5 0.5 0.13 0.5 0.5 1 0.5 0.5 0.5 0.5 /

LOD 3 5 6 6 6 6 6 4 4 7 5 8 5 7 7 /

SD%

P&T/GC/MS

0.9989 0.9949 0.996 0.9975 0.9981 0.9878 0.9878 0.998 0.9966 0.9951 0.9917 0.9969 0.9979 0.9977 0.9967 /

R2

0.2 0.55 0.3 0.75 0.2 0.15 0.15 0.15 0.95 0.7 1.05 0.6 0.25 0.4 0.4 /

LOD

USEPA 8260B

TABLE 16.1 Characteristic Ions (m/z); MIMS and P&T/GC/MS Limit of Detection (LOD, ppb), Standard Deviation (SD %), and R2; USEPA Method 8260B LODs (ppb)

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Practical Aspects of Trapped Ion Mass Spectrometry, Volume V

quantiication functions; and (c) display and archiving of the results in the remote station. Veriication of the status of the device, simple operations related to the control of the mass spectrometer, and checking of the raw results (for example, air/ water checks, tuning, view of the total ion current and mass spectra, etc.) were performed remotely by means of a commercial software package (Laplink Software Inc., Bellevue, WA, USA).

16.3.2

LABORATORY TESTS

Limits of Detection (LOD) were determined by subsequent dilutions of standard solutions down to a signal-to-noise ratio, S/N, of ≥ 3. Signal reproducibility was determined by six replicates of analyte solutions with concentrations ten times larger than the LOD. Finally, linearity was calculated over a concentration range extending from the LOD to 20–100 times the LOD values. All analyses were performed with solutions freshly prepared immediately before use by appropriate dilution of mother solutions with organic-free triply-distilled water. In turn, mother solutions were prepared daily by dilutions of concentrated solutions of the analytes in methanol stored in a refrigerator except during the daily preparation of solutions. MIMS results were compared to those obtained by USEPA Method 8260B based on P&T/GC/MS. A Tekmar Velocity XPT Purge and Trap (Teledyne Tekmar, Mason, OH, USA) coupled to a Varian Star 3400X Saturn 2000 GC/MS (Varian, Palo Alto, CA, USA) was used under the following conditions: P&T Sample volume: 5 mL; Trap: Supelco Trap E (SP 2100/Tenax/Silica gel/Charcoal); Purge temperature: 30°C; Purge time: 11 min; Purge low: 40 mL min−1; Desorbing temperature: 180°C; Desorbing time: 4 min; Desorbing low: 300 mL min−1; Bake temperature: 180°C; Bake time: 10 min; Bake low: 400 mL min−1; Transfer-line temperature: 150°C. GC/MS Column: Supelco SPB 624, 60 m x 0.32 mm ID, 1.8 µm ilm thickness; Injector temperature: 125°C; Oven temperature: from 35 to 50°C at 4°C min–1 holding the initial temperature for 2 min; then to 220°C at 10°C min–1 holding the inal temperature for 10 min; Mass spectra: m/z 25–300 at 1 scan min–1.

16.3.3

FIELD TESTS

Four MIMS instruments were deployed for ield tests in plants that produced water: one instrument was deployed in each of two plants that produced drinking water from

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TABLE 16.2 MIMS Performance in Field Experiments Field Test Site A B C D

Application Ground Water Potabilization Ground Water Potabilization Surface Water Potabilization Industrial Wastewater Treatment

Total

Days On

Analyses /day

Total Analyses

Days Off

% Off

323

11

3

24

7752

492

24

5

48

23,616

510

37

7

24

12,240

526

20

4

3

1587

1,851

92

5 (aver.)

23 (aver.)

45,195

Note: A tabulation of the operational performance of the MIMS instruments with respect to functional days, non-functional days, the percentage of non-functional days, analyses/day, and total number of analyses for each of four sites.

ground water; one instrument was deployed in a plant that produced drinking water from surface water; and the fourth instrument was deployed in a plant for the treatment of industrial waters. All the plants were located in the area near Bologna. The instruments were programed to sample and to analyze water (analysis duration: 5 min; 1 scan per ive seconds full scan of the mass range: m/z 50–200) with the frequency of analysis ranging from three analyses per day to two analyses per hour. Instrument performances were checked over a period ranging from 323 to 526 days (Table 16.2).

16.4 16.4.1

RESULTS AND DISCUSSION LABORATORY TESTS

The compounds used for the present study were chosen on the basis of the following criteria: halomethanes and haloethanes (compounds 8–15 in Table 16.1) are solvents and disinfection by-products; for compounds 8 and 2–4, the sum of the concentrations in drinking water must be less than 30 µg L –1 whereas for compounds 9 and 10, the threshold is 10 µg L –1; the remaining compounds (compounds 1–7 and acrylonitrile, Table 16.1) are of interest because they are often found in industrial wastewaters such as were used for the present study. 16.4.1.1 Detection Limits (LOD) MIMS detection limits (LOD, S/N ≥ 3) were determined by analysis of reference solutions and were compared with (a) LODs obtained by P&T/GC/MS operated as described in the previous section, and (b) LODs reported by USEPA Method 8260B (Table 16.1). MIMS LODs were (a) smaller than those obtained by P&T/GC/MS for the organohalogen compounds and for benzene by about, in some cases, one order of magnitude; (b) comparable to the other technique for toluene, ethylbenzene, styrene,

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250

Counts

200 150 0.03 ppb 100 50 0 10

20

30

40 50 Minutes

60

70

80

90

FIGURE 16.1 A typical example of a temporal trace of the ion current for m/z 130-132 from trichlorethylene at a concentration of 0.03 ppb, showing the signal intensity and S/N ratio at the detection limit.

and for the two dichlorobenzene isomers (compounds 6 and 7); and (c) markedly higher for cumene. Acrylonitrile showed a high LOD probably due to its poor partitioning equilibrium in the membrane that, in turn, can be ascribed to its relatively high polarity. USEPA Method 603 reports 0.5 ppb as the detection limit for acrylonitrile, using Purge and Trap and gas chromatography with either a Porapak or a Chromosorb 101 packed column. Figure 16.1 shows the ion current (m/z 130–132) of a 0.03 ppb solution of trichloroethylene as a typical example of the signal intensity and S/N ratio at the detection limit. 16.4.1.2 Reproducibility The reproducibility (six replicates) of MIMS’ responses was compared to that obtained by the reference method. The results (expressed as standard deviation percentage, SD%, Table 16.1) were comparable for the two methods, with the exception of carbon tetrachloride and bromoform, whose MIMS standard deviations were greater than those obtained by P&T/GC/MS. The standard deviation percentage for compounds of relatively high polarity and/or low volatility (such as toluene, ethylbenzene, cumene, bromoform, and carbon tetrachloride) was relatively higher than that obtained by P&T/GC/MS; these results probably indicate an unfavorable partitioning equilibrium for these particular compounds in the membrane. 16.4.1.3 Linearity With respect to linearity (Table 16.1), MIMS and P&T/GC/MS were comparable (R2 > 0.99), with the exception of cumene, whose MIMS R2 was 0.9087, probably

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due to the previously-mentioned lower partitioning equilibrium of this compound in the membrane; this observation was consistent with the high detection limit of this compound. Acrylonitrile (Figure 16.2) showed perfect linearity over the 50–750 ppb concentration range. 16.4.1.4 Matrix Effects Standard additions of BTEXs to industrial wastewaters showed no matrix effect. The angular coeficient of the straight line obtained by four additions in the 0.1–2 ppm range was practically identical to that of a similar calibration plot using triplydistilled water (18,954 vs 18,883), the two lines being parallel (Figure 16.3).

16.4.2

CASE STUDIES

16.4.2.1 Acrylonitrile Acrylonitrile, a compound that is not included in the family of the VOCs, could be determined at high concentration levels (4.78 ppm) in industrial waters containing

2.0

Ione: 52

750 ppb 500 ppb

kCounts

1.5 1.0

200 ppb

0.5

50 ppb

100 ppb

0.0 25

50

Minutes

75

100

125

FIGURE 16.2 The m/z 52 ion current for acrylonitrile showed perfect linearity over the 50–750 ppb concentration range.

50,000

Signal

40,000 30,000 20,000 10,000 0

0

0.5

1

ppm

1.5

2

2.5

FIGURE 16.3 Calibration of BTEXs in triply-distilled (continuous line, y = 18,883x + 115.59, R2 = 1) and industrial water (dotted line, y = 18,954x + 7002.1, R2 = 0.9977).

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a wide range of aromatic hydrocarbons. Figure 16.4 shows a MIMS full scan mass spectrum with ions at m/z 78, 104, 91, 120, 121 and 118 (typical of such aromatic substances as styrene, ethylbenzene, xylene, cumene, cumene hydroperoxide, α-methylstyrene) along with an ion at m/z 52 (acrylonitrile) of much lower ion signal intensity, whose ion currents for duplicate analyses are shown in Figure 16.5. 16.4.2.2 Comparison of Membrane Inlet Mass Spectrometry (MIMS) with Purge-and-Trap/Gas Chromatography (GC)/ Mass Chromatography (MS) A series of experiments was performed in order to compare MIMS and Head-Space Purge and Trap /GC/MS by analyzing a total of 20 industrial wastewater samples from seven different sampling points. In Figure 16.6 is shown the MIMS mass spectrum of one of the wastewater samples; in this example, seven compounds that

104

Relative intensity

100%

75%

50% 91

25% 51

63

119

78

134

155 165 179

0% 50

100

150 m/z

205 219 200

kCounts

FIGURE 16.4 Full scan mass spectrum of a sample of industrial waters containing acrylonitrile (m/z 52) along with aromatic substances such as styrene, ethylbenzene, xylene, cumene, cumene hydroperoxide, and α-methylstyrene.

25 20 15 10 5 25

Minutes

50

FIGURE 16.5 Ion current of m/z 52, acrylonitrile at 4.78 ppm, duplicate analysis of the same sample of industrial waters as was used for Figure 16.4.

50

55

65 61

60

66

75

Chloroform

79

83

100

105

Toluene 91 101 1,2 dichloroethylene 96 CCl4 117 98

125 m/z

130

134

Trichloroethylene 132

150

151

175

Tetrachloroethylene 166

200

FIGURE 16.6 Mass spectrum of industrial water used to compare MIMS and Purge-and-Trap/GC/MS. Identiied compounds and ions used for quantiication are reported.

0%

25%

50%

75%

100%

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y = 1,0671x – 0,6296 R 2 = 0,8944

ppb (P&T/GC/MS)

25,00 20,00 15,00 10,00 5,00 0,00

0

–5,00

5

10

15

20

25

ppb (MIMS)

FIGURE 16.7 Comparison of data obtained by analyzing samples with Purge-and-Trap/ GC/MS and MIMS. The concentration (in ppb) of each of toluene (m/z 91), benzene (m/z 78), 1,2-dichloroethylene (m/z 98), trichloroethylene (m/z 130 + 132), chloroform (m/z 83), and vinyl chloride and dichloroethane (m/z 62 for both compounds) in each of 20 wastewater samples determined by MIMS is plotted against that determined by Purge-and-Trap/GC/MS.

have been identiied by their characteristic ions are indicated on the mass spectrum. Toluene (m/z 91), benzene (m/z 78), 1,2-dichloroethylene (m/z 98), trichloroethylene (m/z 130 + 132), chloroform (m/z 83), and vinyl chloride and dichloroethane (m/z 62 for both compounds) were detected and quantiied in all 20 samples with both techniques. The concentration (in ppb) of each of the above seven compounds in each of 20 wastewater samples determined by MIMS is plotted against that determined by P&T/GC/MS as shown in Figure 16.7. The equation of the regression line calculated from these data (Figure 16.7) is y = 1.0671x–0.6296, R2 = 0.8944. The slight differences between the actual and the ideal coeficients (slope = 1, intercept = 0 and R2 = 1) are probably due to the contributions of other compounds to the abundances of the ions used for quantitation.

16.4.3

FIELD TESTS

Four instruments were deployed in different plants representative of typical cases of water treatment, namely two plants for the potabilization of ground water (A and B), the third plant for surface water potabilization (C), and, inally, a plant for industrial water treatments (D) (Table 16.2). In ield test A, the instrument was deployed in a plant for the production of drinking water from ground water using chlorine dioxide as a disinfection agent. Due to past industrial activity in that area, the ground water was heavily contaminated by chloroform and trichloroethylene. Charcoal ilters were used to abate the organohalogen concentration in drinking water down to 1–10 ppb levels. The instrument was located in a 2 × 3 m container maintained at room temperature. The instrument was able to identify and to quantify drinking water pollutants by means of a mass spectrum in which the ions characteristic of the individual compounds were recorded clearly, showing their

Remote Monitoring of Volatile Organic Compounds in Water

503

respective diagnostic isotopic patterns (Figure 16.8a). Hourly analyses were carried out in order to check that pollutant concentrations did not exceed the legal thresholds. The position was unmanned and the results were transmitted to the remote control room by e-mail at the conclusion of each analysis. The instrument was monitored over a period of 334 days during which it functioned for 323 days; failures and maintenance resulted in the loss of 11 days (that is, 3% of the time monitored) (Table 16.2). Almost 8000 determinations were performed corresponding to 646 hours of analysis. In plant B, both ground water and drinking water were monitored every hour, corresponding to a total frequency of one analysis per 30 minutes. Here, the contaminant was trichloroethylene. The plant was not equipped with charcoal ilters, consequently the pollutant concentration was kept within the regulation limit (10 ppb) by shifting water uptake from one well to another. Figure 16.8b shows a typical full-scan mass spectrum recorded from the ground water of this location, with the characteristic trichloroethylene molecular ion isotopic quartet at m/z 130, 132, 134, and 136. As for the previous ield study in plant A, the location was unmanned and the results were transmitted by e-mail. The percentage of inactivity in plant B (5%) was comparable with that of plant A (3%), despite the fact that the working period for plant B (almost 500 days) was longer than that for plant A, and the number of analyses carried out at plant B (23,626) was more than three times higher than those carried out in plant A (7,752); see Table 16.2. Field test C was an example of application to surface waters used for human consumption. Such waters were essentially uncontaminated by chemicals and needed only a conventional disinfection. Nevertheless, this plant was monitored in consideration of the fact that accidental pollution by gasoline and oil had been recorded in the past due to the proximity of an adjacent highway with heavy trafic. Figure 16.8c shows a typical mass spectrum of this instance, with no signiicant ions. This instrument was monitored over 547 days (12,240 analyses, 1020 hours) during which the days off were 37, that is 7% of the period (Table 16.2). Finally, industrial wastewaters (plant D) were analyzed from the outlet of a pipe connected into a municipal sewage treatment plant. In such a case, the concern was that organohalogenated compounds from industrial wastes may affect the biological treatment of urban wastewaters. The instrument recorded 3 analyses per day on those days when the industrial wastewaters were discharged. Figure 16.8d shows the typical mass spectrum of such samples. Ions characteristic of chloroform (m/z 83, 0.3 ppb), trichloroethylene (m/z 130, 23 ppb), and toluene (m/z 91, 31 ppb) were found in the mass spectrum reported in Figure 16.8d. The complex matrix of such wastes did not affect the MIMS determinations (see previous discussion, Figure 16.3). The instrument was monitored during more than 500 days (about 1600 analyses or 130 functioning hours) with a 4% of non-functioning time (Table 16.2). For all types of mass spectrometers, particularly those operated in remote locations, it is of interest to consider the frequency of mass calibration and, for those instruments that employ electron impact ionization, the frequency with which the ilament must be replaced. It was found for the MIMS instruments that mass calibrations were carried out on 24 occasions and ilaments were replaced on 16 occasions.

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(a) 100%

164

75% Chloroform 94 96 83

50%

166 Tetrachloroethylene 168

25% 0% 50

75

100

125

150

175

m/z

(b) 100%

130 132

75% Trichloroethylene

60 50% 96

134

100

150 m/z

25% 0% 50 (c)

100%

250

45 52

75% 50%

200

41

25% 0% 50

75

100

(d)

125 m/z

150

175

200

91 Toluene

100% 75% 50%

Chloroform

25%

77 83

105

Trichloroethylene 130 132

0% 50

100

m/z

150

200

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FIGURE 16.8 (Opposite) (a) Field test A. Mass spectrum of a water sample from a plant for the production of drinking water from ground water. Chlorine dioxide had been used as a disinfection agent. Ground water was contaminated heavily by chloroform and trichloroethylene due to past industrial activity; charcoal ilters had been used to abate the organohalogen concentration in drinking water down to 1–10 ppb levels. (b) Field test B. Mass spectrum recorded from the ground water of this location. At this site, the contaminant was trichloroethylene and the mass spectrum shows the characteristic trichloroethylene molecular ion isotopic quartet at m/z 130, 132, 134, and 136. The plant was not equipped with charcoal ilters, consequently pollutant concentration was kept within the regulation limit (10 ppb) by shifting water uptake from one well to another. (c) Field test C. Mass spectrum of suricial water used for human consumption. Such waters were essentially uncontaminated by chemicals and needed only a conventional disinfection. No signiicant ions were observed. (d) Field test D. Mass spectrum of a sample of industrial wastewaters taken from the outlet of a pipe connected into a municipal sewage treatment plant. In this case, the concern was that organohalogenated compounds from industrial wastes may affect the biological treatment of urban wastewaters. This typical mass spectrum shows ions characteristic of chloroform (m/z 83, 0.3 ppb), trichloroethylene (m/z 130, 23 ppb), and toluene (m/z 91, 31 ppb).

From the data shown in Table 16.2 concerning the numbers of operating days and the number of analyses carried out each day at each of the four sites, it is found that averages of 2825 analyses were carried out with each ilament and 1883 analyses were carried out between successive calibrations. Comparing these data with those of a GC/MS instrument used presently in our laboratory and which has shown good instrumental stability and reliability, it was found that the GC/MS instrument performed 305 analyses per ilament and 78 analyses per calibration. When it is borne in mind that the duration of a GC/MS analysis was 50 min while that of a MIMS analysis was 5 min, it is clear that both systems performed comparably in terms of operation time per ilament.

16.5

CONCLUSION

A number of laboratory tests to determine LOD, linearity and repeatability of MIMS instruments applied to the analysis of VOCs in water were performed. Data were comparable with those obtained by the classical method of VOC analysis in water (P&T/GC/MS and USEPA Method 8260B). Four MIMS instruments were tested over an extensive period of time to evaluate their on-site performance in unmanned locations. Results were remarkable: the instruments worked unchecked for long periods producing a total of more than 45.000 analyses and VOC amounts were quantiied automatically and sent to a remote control room where non-expert personnel could understand the results readily. In conclusion, MIMS instruments proved to have great potential for utilization in continuous VOC-monitoring stations. These instruments are reliable, cost-effective and simple to use; they have no environmental impact because no solvent is used for the extraction of organics from water, and they can be located on-site, unattended, providing a continuous low of data on water quality and pollution.

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REFERENCES 1. Irvine, K.N.; McCorkhill, G.; Caruso, J. Continuous monitoring of conventional parameters to assess receiving water quality in support of combined sewer overlow abatement plans. Water Environ. Res. 2005, 77, 543–552. 2. Mikkelsen, Ø.; Skogvold, S.M.; Schrøder, K.H. Continuous heavy metal monitoring system for application in river and seawater. Electroanalysis 2005, 17, 431–439. 3. Yamada, M.; Suga, M.; Waki, I.; Sakamoto, M.; Morita, M. Continuous monitoring of polychlorinated biphenyls in air using direct sampling APCI/ITMS. Int. J. Mass Spectrom. 2005, 244, 65–71. 4. Hart, K.J.; Dindal, A.B.; Smith, R.R. Monitoring volatile organic compounds in lue gas using direct sampling ion trap mass spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 352–360. 5. Kürten, A.; Curtius, J.; Helleisa, F.; Lovejoy, E.R.; Borrmann, S. Development and characterization of an ion trap mass spectrometer for the on-line chemical analysis of atmospheric aerosol particles. Int. J. Mass Spectrom. 2007, 265, 30–39. 6. Riter, L.S.; Laughlin, B.C.; Nikolaev, E.N.; Cooks, R.G. Direct analysis of volatile organic compounds in human breath using a miniaturized cylindrical ion trap mass spectrometer with a membrane inlet. Rapid Commun. Mass Spectrom. 2002, 16, 2370–2373. 7. Johnson, R.C.; Srinivasan, N.; Cooks, R.G.; Schell, D. Membrane introduction mass spectrometry in a pilot plant: On-line monitoring of fermentation broths. Rapid Commun. Mass Spectrom. 1997, 11, 363–367. 8. Bier, M.E.; Cooks, R.G. Membrane interface for selective introduction of volatile compounds directly into the ionization chamber of a mass spectrometer. Anal. Chem. 1987, 59, 597–601. 9. Kotiaho, T.; Lauritsen, F.R.; Choudhury, T.K.; Cooks, R.G.; Tsao, G.T. Membrane introduction mass spectrometry Anal. Chem. 1991, 63, 875A–883A. 10. Lauritsen, F.R.; Kotiaho, T.; Choudhury, T.K.; Cooks, R.G. Direct detection and identiication of volatile organic compounds dissolved in organic solvents by reversedphase membrane introduction tandem mass spectrometry. Anal. Chem. 1992, 64, 1205–1211. 11. Bauer, M.; Solyom, D. Determination of volatile organic compounds at the parts per trillion level in complex aqueous matrixes using membrane introduction mass spectrometry. Anal. Chem. 1994, 66, 4422–4431. 12. Soni, M.; Bauer, S.; Amy, J.W.; Wong, P.; Cooks, R.G. Direct determination of organic compounds in water at parts-per-quadrillion levels by membrane introduction mass spectrometry. Anal. Chem. 1995, 67, 1409–1412. 13. Cisper, M.E.; Gil, C.G.; Townsend, L.E.; Hemberger, P.H. Online detection of volatile organic compounds in air at parts-per-trillion levels by membrane introduction mass spectrometry. Anal. Chem. 1995, 67, 1413–1417. 14. Mendes, M.A.; Pimpim, R.S.; Kotiaho, T.; Eberlin, M.N. A cryotrap membrane introduction mass spectrometry system for analysis of volatile organic compounds in water at the low parts-per-trillion level. Anal. Chem. 1996, 68, 3502–3506. 15. Ketola, R.A.; Mansikka,T.; Ojala, M.; Kotiaho, T.; Kostiainen, R. Analysis of volatile organic sulfur compounds in air by membrane inlet mass spectrometry. Anal. Chem. 1997, 69, 4536–4539. 16. Bocchini, P.; Pozzi, R.; Andalò, C.; Galletti, G.C. Membrane inlet mass spectrometry of volatile organohalogen compounds in drinking water. Rapid Commun. Mass Spectrom. 1999, 13, 2049–2053.

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17. Ketola, R.A.; Kotiaho, T.; Cisper, M.E.; Allen, T.M. Environmental applications of membrane introduction mass spectrometry. J. Mass Spectrom. 2002, 37, 457–476. 18. Srinivasan, N.; Johnson, R.C.; Kasthurishnan, N.; Wong, P.; Cooks, R.G. Membrane introduction mass spectrometry. Anal. Chim. Acta. 1997, 350, 257–271. 19. Bocchini, P.; Pozzi, R.; Andalò, C.; Galletti G.C. Experimental upgrades of membrane introduction mass spectrometry for water and air analysis. Anal Chem. 2001, 16, 3824–3827.

Author Index* Ausio, J., 69 Abedi, A., 408, 409 Adamczyk, M., 88, 90 Amunugama, M., 91 Arriaga, E.A., 99 Back, J.W., 104 Badman, E.R., 8, 17 Baessmann, C., 282 Bagal, D., 227 Bartlet-Jones, M., 98 Bateman, R.H., 210 Berton. A., 367 Bier, M.E., 447 Bisgaard, C.Z., 312 Blatt, R., 359 Blom, M.N., 180 Bocchini, P., 491 Bowers, M.T., 207, 208, 219 Brancia, F.L., 367 Brekenfeld, A., 282 Brittain, R., 445 Brock, A., 140 Brodbelt, J.S., 36, 39, 46–48, 55 Bruce, J.E., 104 Burns, M.M., 180 Bush, M.F., 244, 248, 249 Caldwell, G., 397, 399–402 Carter, J.G., 408 Champenois, C., 333 Chen, X., 103 Chipuk, J.E., 36, 39, 46, 48, 55, 65 Chowdhury, S., 410 Chrisman, P.A., 8, 13 Christophorou, L.G., 408 Chung, S., 354 Clemmer, D.E., 216 Clench, M., 225 Cooks, R.G., 170, 224, 278, 328 Coon, J.J., 10, 22, 59, 63, 65, 69, 71, 72 Cooper, H.J., 121, 207 Cudzilo, K., 434 Danell, R.M., 180 Daniels, S., 98 Dehmelt, H.G., 170, 328, 334 Dey, S., 98 Dick, G.J., 337 Djdja, M.-C., 226

Douglas, D.J., 52, 53, 380, 381 Drewsen, M., 254, 291, 294, 296, 297, 300, 309, 311, 312, 356 Drexler, D.M., 435 Dryhurst, D.D., 69 Duft, D., 189, 190, 193, 194, 196, 197 Dunbar, R.C., 248, 249 Eckers, C., 228 Eiceman, G.A., 206, 387, 404 Erickson, D.E., 13 Evoy, S., 309 Ewing, R.G., 404, 405 Fenn, J., 127 Fico, M., 328 Fitaire, M., 396, 397 Fohlman, J., 131 Forbes, M.W., 239, 248, 249 Franzen, J., 263, 264, 277, 282 Froelich, J.M., 60, 83 Galletti, G.C., 491 Gao, L., 328 Gardner, M.W., 109 Garrett, T.G., 225 Garzón, I.L., 179, 180, 417, 420, 428, 433 Gauthier, J.W., 132 Ge, Y., 144 Gebler, J.C., 90 George III, J.E., 439 Gerlich, D., 245, 335 Gheno, F., 396, 397 Giles, K., 210 Glish, G.L., 6, 70, 465 Goshe, M.B., 105, 106 Grabenauer, M., 219 Grifin, T.J., 99, 100 Grimsrud, E.P., 397, 399–402, 410 Gronert, S., 43 Gunawardena, H.P., 70 Gygi, S.P., 94, 142 Haberland, H., 171 Hakansson, K., 140 Han, H., 23 Han, H.L., 64, 66 Harden, C.S., 404, 405 Hartmer, R., 282 Harvey, D.J., 227 Hattan, S., 98

* The names listed here refer only to authors whose names appear in the text and/or in the captions.

509

510 He, F., 98 Hilton, G.R., 219 Hiraoka, K., 397 Hogan, J.M., 18 Højbjerre, K., 254, 291, 311, 312, 356 Holland, R., 227 Huang, Y.N., 98 Hunt, D.F., 10, 22, 36, 37, 63, 69, 139, 451 Hunter, E.P., 38 Hunter, S.R., 408 Iavarone, A.T., 189, 190 Jackson, G.P., 463 Jacobson, A., 98 Jensen, L., 296, 297 Jockusch, R.A., 239, 248, 249 Johnson, R.C., 493 Juhasz, P., 98 Julien, R.R., 93 Julka, S., 94, 95 Jung, H.R., 228 Kaplan, D., 282 Karpas, Z., 206 Kebarle, P., 395, 402, 410 Ketola, R.A., 493 Khainovski, N., 98 Kiessel, S.E.B., 71, 72 Kim, S.H., 395 Knighton, W.B., 402, 410 Konenkov, N.V., 380, 381 Kürten, A., 493 Landman, U., 181, 182 Laskin, J., 140 Lawless, P.A., 406 Lawrence, A.H., 402 Le, T., 354 Li, L., 85 Li, S., 97 Liang, X.R., 11, 13 Lias, S.G., 38 Lindballe, J., 296, 297 Liu, J., 3, 13, 64 Liu, Y., 408 Londry, F.A., 13 Louris, J.N., 459 Lu, Y., 60, 83 Ly, T., 93 Magnera, T.F., 402 Maleki, L., 337, 340, 354 Manura, D., 273 Mao, D., 52 March, R.E., 350, 440 Marchese, J.N., 98 Margolis, H., 346 Marshall, A.G., 130, 132, 138, 144 Martin, S., 98 Martinussen, R., 294, 296, 297, 300, 309 Mayhew, C.A., 407, 408

Author Index McAlister, G.C., 59, 71, 72 McEwen, C.N., 225 McLean, J.A., 225 McLuckey, S.A., 3, 7, 8, 11, 13, 17, 18, 20, 23, 62, 64, 66–68, 72 Meany, D.L., 98, 99 Michaelian, K., 179, 180 Mikkelsen, Ø., 492 Mordehai, A., 440 Mortensen, A., 294, 296, 297, 300, 309, 312 Mulholland, J.J., 464 Mulligan, C.C., 328 Newton, K.A., 20 Nissen, N., 296, 297 Offenberg, D., 312 Olivova, P., 226 Oomens, J., 248, 249 Ouyang, Z., 328 Pappin, D.J., 98 Paradisi, C., 371 Parker, K., 98 Parks, J.H., 169, 180, 181, 189, 190, 193, 194, 196, 197 Paul, W., 262, 328, 439 Payne, A.H., 70 Peverall, R., 408 Pillai, S., 98 Pinelli, F., 491 Plass, W.R., 275–277 Plet, B., 153 Polfer, N.C., 248, 249 Pozzi, P., 491 Prestage, J.D., 337, 340, 354 Preston, J.M., 396, 398 Przybylski, M., 140 Purkayastha, S., 98 Purves, R.W., 209 Qiu, Y., 96 Rajadhyax, L., 396, 398 Ramsey, N., 332 Raveane, L., 367 Regnier, F., 94, 95 Reich, R.F., 434 Reid, G.E., 17, 60, 83, 91, 100 Reilly, J.P., 252 Riba-Garcia, I., 226 Ridenour, W.B., 225 Rizzo, T.R., 245 Roberts, K.D., 91 Roepstorff, P., 130, 131 Ross, P.L., 98 Rutherford, E., 3 Sadagopan, N., 89 Sahlstrom, K.E., 409, 410 Schmitter, J.-M., 153 Schrama, C.A., 346 Schroeder, M.J., 10, 22, 63

511

Author Index Schubert, M., 282, 440 Schwartz, J.C., 65, 440, 461, 463–465 Scrivens, J.H., 205, 219 Shabanowitz, J., 10, 22, 63, 69 Shi, X., 193, 194, 196, 197 Simon, C., 155, 164 Slade, S.E., 219 Smith, R.D., 132, 138 Soderblom, E.J., 105, 106 Song, Q., 328 SØrensen, J.L., 294, 300, 309 Spangler, G.E., 406 Specht, A., 440 Staanum, P.F., 254, 291, 294, 296, 297, 300, 309, 311, 312, 356 Stapelfeldt, H., 312 Stauber, J., 226 Steinwedel, H., 439 Stephenson J.L., 7, 63, 67, 68 Stick, D., 295 Stone, J.A., 206, 387, 404 Strife, R.J., 449 Sudakov, M., 380, 381 Summerield, S.G., 89 Swaney, D.L., 64 Syka, J.E.P., 10, 22, 63, 65, 69 Tabrizchi, M., 408, 409 Talbot, F.O., 239 Tanaka, K., 127 Taylor, D.M., 444, 459, 463 Thalassinos, K., 205, 219, 223 Thompson, L.V., 99 Thompson, R.C., 334 Thomson, J.J., 3 Tjoelker, R.L., 340

Todd, J.F.J., 350, 440 Traldi, P., 367 Trim, P.J., 225 Turecek, F., 136 Ueberheibe, B., 69 Uetrecht, C., 221 Vedel, F., 327 Voight, D., 296, 297 Wang, H., 86 Wang, M., 445 Wang, N., 85 Watson, J.T., 89 Wells, G.J., 447, 462 Wells, J.M., 17 Wester, R., 311 Williams, E.R., 248, 249 Williams, J.P., 228 Williamson, B., 98 Wineland, D., 170 Wirtala, M., 65 Wright, P.J., 53 Wu, J., 90 Xia, Y., 11, 23, 62, 66 Xie, H., 99 Xing, X., 180, 181 Yamabe, S.J., 397 Yamada, M., 492 Yang, M., 23, 439 Yang, M.J., 13 Yoon, B., 181, 182 Yost, R.A., 225, 417, 434, 446, 464 Zeng, D., 97 Zhang, J., 47, 53 Zhou, H., 96 Zubarev, R.A., 143

Subject Index 1,4-Dinitrobenzene, 399 1,1,1-Trichloroethane, 496 1,2-Dichloroethylene, 501, 502 1,2-Dichlorobenzene, 495 1,2-Dichloroethane, 492 1,4-Dichlorobenzene, 495 115In +, 359 171Yb +, 345 18O atom, 103 18O −, 71 2 199Hg+, 345, 352, 356 2-(2′-Hydroxybenzoyl)–benzoic acid, 371, 372 2,2′,4,4′,6-Pentabromodiphenylether, 480 2,3-Dichlorobiphenyl, 484, 485 2,3-Dimethyl pyridine, protonated, 391 2,3-Dimethyl-2,3-dinitrobutane, 399, 400 2,3-Dimethyl-2,4-dinitropentane, 399 2,4-Dichlorophenol, 472–474 2,4-Dimethylpyridine, 403–406 2,4-Dinitroluorobenzene-d 0/d3, 103 2,5-Hydroxybenzoic acid, 425, 428, 429 cluster ion of, 430 2,6-Naphthalic acid, 48 202Hg+, 352 24Mg+, 307, 312, 324 24MgH+, 324 25Mg+, 324 2D Polyacrylamide gel electrophoresis, 2D PAGE, 93 2D Quadrupole ion trap, 417, 419 2-Deoxy-5-cytidine monophosphate, 46 2-Methoxy-4,5-dihydro-1H-imidazole, 88 3-(3-Methoxypropoxy) propanol, 396 3,3′-Dithio-bis(succinimidylpropionate), DTSSP, 104 3D Quadrupole ion trap mass spectrometer, 5, 9, 7, 17, 51, 54, 62, 242, 257, 282, 417, 419, 440 40 Ca +, 300, 305, 307–309, 312–316, 318, 320, 321 40 Ca16O +, 300, 305, 310 42Ca +, 309 44Ca +, 309 4-Sulfophenyl isothiocyanate, 89 5′P-dAA, 55 5′P-Dag, 55 5′P-dGA, 55 5′P-dGG, 55 63Ni source, 207, 390, 395

6-Aza-2-thiothymine, 431, 433 Sr+, 345, 359

88

A Absorption spectroscopy, 240 AC dipole electric ield, 440 Accidental contamination, 492 Accurate mass tag, AMT, 129, 140 Acetic acid, 127 Acetone, 396 Acetone–water, 397 Acetonitrile, 127, 155, 481 Acetylacetone, 88 Acid-labile isotope-coded extractants, ALICE, 96 Acquisition phase, 372 Acrylonitrile, 491, 495, 498–500 Acrylonitrile butadiene styrene plastic, ABS, 480, 482 Action spectroscopy, 240, 246, 247, 253, 282 Activated ion electron capture dissociation, AI-ECD, 137 Activation barrier, 371 Activation energy, reverse, 496 Adduct ions, 411 dissociation of, 387, 403 Adiabatic approximation, 349 Adiabatic cooling, 396 Afinity capture method, 86 Afinity tag, 105 Aging, 425 Agn+ cluster, 171, 174, 177, 178–181 Al2O3, 86 Al3+, 86 Alcohol dehydrogenase, 141, 219 Aldolase, 219 Allan deviation, 331 Allan Variance, 331 Alveolar proteomics, 140 cystic ibrosis, 140 proteinosis, 140 Alzheimer’s disease, 220 Ambient pressure, 387, 389 American Society for Mass Spectrometry, 284 Amino acid residue, modiication site, 84 Ammonium acetate, 127 Ammonium hydroxide, 127 Amplitude detection, 304, 310, 320

513

514 Amplitude method, 304, 320, 321, 323 Amplitude, modulation frequencydependent, 304 Amyloid ibril, 220 Amyloidogenic protein β2-microglobulin, 220 Analysis, drug, 441 food, 441 forensic, 441 Analytical mass scan, 375, 377 Anharmonic bottleneck, 250 Anharmonicity, 315, 316, 338 Aniline ion (C6H5NH2+), 311 photofragmentation, 311, 312 Anion formation, 408 Anthracene, 452, 453, 455 Anthropic activities, 492 Apigenin, 155, 156, 162, 163 Apigenin-7-O-glucoside, 155, 156, 162, 163 Apigenin-7-O-neohesperidoside, 155, 156, 162, 163 Apomyoglobin, 64 Applied Biosystems peptide synthesizer, 156 Arabidopsis, 65 Arginine, 24, 127, 191, 247–249 Argon, 244, 368 Arrhenius parameters, 410 Arrhenius plot, 405, 409 Arrhenius rate model, 197, 198 Arrival time, 401 corrected, 218 distribution, 212 distribution proile, 214, 217, 223 ASGDI, 6, 7 ASGDI source, 8, 11 Aspartic acid cleavage, 16 Aspartic acid residue, 22 location of, 90 Astragalin, 50 Astronomical time, 329 Atmospheric pressure, 388 chemical ionization, APCI, 8, 12, 492 ion emitter, dual, 63 solids analysis probe, ASAP, 225 Atmospheric sampling glow discharge ionization, see ASGDI Atomic beam, 332, 342 Atomic clock, 327–360 schemes, 332 Atomic fountain, 331, 332 Atomic ion lifetime, 328 Atomic ion, laser-cooled, 292–295, 297, 299, 300, 304, 305, 310, 317, 318, 323 Atomic ions, 170, 299 luorescence, 170 laser cooling, 170, 328 Atomic laser, 328 Atomic levels, 358

Subject Index Atomic oscillator, 331, 360 Atomic quantum physics, 328 Atomic spectroscopy, high resolution, 328 Atomic transition, 330, 331, 335, 339, 355 Atrazine, 478, 479 Attachment rate coeficients, 407 au, 261 AuCl2−, 70, 175, 176, 182–186 Aun− cluster, isomer structure, 184–186 Automated method development, AMD, 467, 486 Automatic gain control, AGC, 422–425, 444, 467 Auxiliary dipolar AC potential, 272 Auxiliary RF voltage, 9, 11 Average dipole orientation, ADO, 402 Averagine, 129 Aviation security, 129 Avidin afinity chromatography, 97 Axial modulation, 272, 273, 440, 456, 467 az, 261, 270, 298, 350, 369 Azobenzene radical anion, 23 Azulene anion, 410

B Ba+, 345 Backbone cleavage, 24 Background gas, 319, 339 Background noise, 447 BAD, see Boundary-activated dissociation BAPMPS, 92 Barium, 332, 334 Baseline interferometry, 330 Bath gas, 5; see also Buffer gas atomic/molecular weight, 446 helium, 42, 43, 171, 174, 175, 195 neon, 175 pressure, 10 temperature, 191 BDE 100, 481 BDE 153, 481 BDE 154, 481 BDE 183, 481 BDE 205, 481 BDE 209, 480–482 BDE 28, 481 BDE 47, 481 BDE 99, 481 Beaker proile, 335 Benzene, 36, 492, 495, 502 Benzoic acid anions, 68–70 Benzyl cation, 104 Beryllium, 319, 357 Biological molecule, 246 identiication, 84, 128 structural characterization, 84, 128

515

Subject Index Biomolecular conformation, 170 Biomolecular ions, 192 trapped, 169 Biomolecule analysis, 15 Biomolecule conformational change, 186 Biomolecule folding, 255 Biomolecule protonated, photo-excitation of, 240 Biomolecule, dye-derivatized, 186–188 Biphenyl, 483, 484 BIRD, see Blackbody infrared radiative dissociation Bisuccinimidyl-succinamyl-aspartyl-proline, SuDP, 106 Bisuccinimidyl-succinamyl-aspartyl-prolylglycine, SuDPG, 106 Black hole (or canyon), 263, 348, 350–352, 353 Blackbody heating, 41 Blackbody infrared radiative dissociation, BIRD, 123, 130, 246, 253 principles, 134 protein and peptide, 134, 135 Blackbody radiation, 41, 332, 359 BMS-X, 435 Body-centered cubic (bcc) symmetry, 177 Boltzmann distribution, 388 Boltzmann sigmoidal function, 158 Boltzmann’s constant, 207 Bottom-up approach, see Proteomics, bottom-up, 101 Boundary activation, 367, 369, 382 Boundary effect, 371 Boundary-activated charge-separation dissociations, 383 Boundary-activated dissociation, 367, 369, 370, 373, 374, 385 Bovine cytochrome c charge states, 218 Bovine serum albumin, 16, 17, 141, 154 Bradykinin, 51, 52, 154 fragment (residues RPPGF), 380, 381 doubly-protonated molecule of, 378, 380, 382–384 Bragg diffraction peak, 177, 178 Brain natriuritic peptide (BNP-32), 140 BrCH2COC6H5, 91 Breakdown curve, 159, 160 Breast carcinoma cell, proteomics analysis of, 140 Breath, human, 493 Breathing (BR) mode, 294, 301–303, 305, 307, 308, 313–316, 322, 323 Brewster angle window, 187 Broad-band frequencies, 369 Bromide ion, 411 Bromodichloromethane, 492 Bromoluorobenzene, 467 Bromoform, 492, 495, 498

Broth, fermentation, 493 Brownian motion, 392 Bruker Daltonics HCTultra, 14 Bruker HCTultra/Agilent 6340 ETD ion trap, 8 Bruker ion trap, 442 BTEX compounds, 492, 499 Buffer gas, 205, 207, 245, 278, 279, 281, 310, 368, 382, 440; see also Bath gas atomic/molecular weight, 446 collision, 335, 354 pressure, 281, 446 Butanedione, 101

C C2D2, 312 C2H5+, 449 C2H5OD, 37 C60 cluster ion, 178, 179 C6D5OD, 38 C6H5CH2OD, 38 Ca+, 295, 299, 318, 349, 350, 355, 358, 359 isotope combination, 309 Cage, miniaturization of, 328 Calcium acetate, 20 Calcium beam, 318 Calibration procedure, 216, 217 California Institute of Technology, 329 Camera, 420 Canada Research Chairs Program, 284 Canadian Foundation for Innovation, 284 Cancer, 418 CaO+, 305 Carbon tetrachloride, 408, 409, 495, 498 Carbonic anhydrase, 133, 138 Carcinogens, 483 Case Studies, 499 Casein, 154 Catechin (+), 49, 155, 156, 162–164 galloylated, 49 non-galloylated, 49 Cation adducts, 421 Cation/anion complex formation, 20 Cationic salts, 421 Cavity ring-down spectroscopy, CRDS, 240, 241 CCD camera, 294, 295, 300, 301, 305, 319–321 image, 180, 310 pixel value, 178 CD3OD, 38, 43, 51–54 Center-of-mass (COM) mode, 294, 301–303, 305, 307–309, 313–316, 319–323 Central barrier, 402 Cerebral peduncle, 425 Cerebrospinal luid, 85 Cesium atom, 329 Cesium atomic frequency standard, 329, 331, 352

516 CF3CH2OD, 38 CH3OD, 38 CH5+, 449 Champagne lute proile, 335 Charge capacity, 442 extended, 439, 442 ion trap, 444 Charge inversion, 12, 15, 19 reaction, 72 negative-to-positive, 19 positive-to-negative, 19 Charge limit, spectral, 444 Charge reduction, 16 stepwise, 19 Charge state, 15, 17, 19, 67 deconvolution, 70 manipulation, 6, 15, 16 Charge transfer, exothermicity of, 197 photoinduced, 196 Charge-coupled device, CCD, 176, 177 Charge-dependent dissociation, 16 Chemical background, 436 Chemical derivatization, 83, 85, 92 reagent, 101 strategy, 88, 89, 91, 94 ixed-charge, 101 Chemical distributions, intrinsic, 418 Chemical ionization reagents, liquid, see Ionization, chemical, liquid Chemical ionization source, 9–11, 14 Chemical ionization, see CI Chemical ionization, see Ionization, chemical Chemical mass shift, 276 Chemical modiication technique, 102 Chemical reaction path, 328 Chemical signature, 418 Chemical structure elucidation, 450 Chemical vapor deposition, CVD, 445 Chemicals Warfare Convention, 229 Chemistry analysis, 328 Chicken egg white proteome, 142 Chloride ion, 400, 401, 403, 406, 407, 410, 411 Chlorine dioxide, disinfection by, 505 Chlorobenzene, 407 Chlorodibromomethane, 492 Chloroform, 408, 492, 495, 501, 502, 504 counter low of, 398 Chloropyriphos, 477, 478 Choline, 425 Chromatographic separation, 85 Chromatographic timescale, 24 Chromosorb 101, 498 CI, see Ionization, chemical CI/MS/MS, see Ionization, chemical/tandem mass spectrometry CID, see Collision-induced dissociation CID-MS/MS, 88, 100

Subject Index Circular dichroism spectroscopy, 219 Cleavable isobaric labeled afinity tag, CILAT, 97 CLIO, 247 Clock atomic transition, 360 Clock signal, 330, 331, 339 building, 341 Clock transition, 343 Cluster ion, 175, 190, 395 symmetry, 182 (CsI)nCs+, 177, 178 Cluster ions, mass-selected, 176 CO, 310 Coating methods, 421 acoustic wave, 421 airbrushing, 421 electrospraying, 421 inkjet, 421 sublimation, 421 Coating, chromium, 445 Coating, Silchrom, 445 Cocaine, 434 Cocaine-d3, 434 Coherent motion, 125 Collision cell, 9 octopole, 208 Collision cross-section, 207, 208, 223 theoretical, 207 Collision energy, 464 normalized, 157–159 Collision frequency, 389 Collision gas, 246 Collision model, hard-sphere, 216 Collision, ion/neutral, 319 Collisional cooling, 278 Collisional dissociation, 392 Collision-induced dissociation, 18, 21, 25, 41, 49, 59–61, 64, 66, 67, 70–72, 90, 92, 96–98, 106, 108, 122, 130, 155–158, 208, 212, 226, 246, 367, 392, 458, 462, 464 beam-type, 23 chemical structure-insensitive, 465 consecutive/competing, scan function for, 373 eficiency, 465 fragmentation eficiency, 66 low energy, 104, 107 multi-level, 464 period, 464, 465 techniques, HASTE CID, 465, 466 techniques, HighQ Pulsed CID, 465, 466 Collisions, thermalizing, 389 COM mode resonance frequency, 300, 301 Combinatorial ligand library bead, 86 Compensation electrode, 346, 347 Compensation voltage, CV, 209, 347

517

Subject Index Complex mixture analysis, 15 Complex, covalently-bound, 71 Concentration gradient, 392 Concentration, maximum allowable, 492 Coninement potential, 335 Conformational dynamics, 170 Conformational family, 219, 223 Conformational luctuations, 186, 191, 195 peptide, 195, 198 Conformational state, 220 Conformer luctuations, rate of, 197, 198 Conformer structure, 195 Congener(s), 480, 481, 484 Contact (or patch) potential, 339, 350 Containment lenses, 12 Contaminated extract analysis, 470 Continuous monitoring system, 492 Continuous wave (CW) laser, 188 Controlled substances, monitoring of, 388 Conversion dynode, 452 Cooling rate, 312 Cooling time, 382 Cooling, 377; see also Laser cooling Copper, oxygen-free high conductivity, OFHC, 337, 338 Corona discharge ionization, 8 Corpus callosum, 423, 424 forceps major of, 425 Correction factor Lz, 349, 350 Correlated harmonic excitation ields, CHEF, 131 Correlated sweep excitation, COSE, 131 Correlation method, 344 Coulombic attraction, long-range, 24 Coulombic explosion, 127 Coulombic force, 316 Coulombic interaction, 294, 295, 299, 302, 307, 313–315, 320, 323, 335, 337 Coulombic repulsion, 241, 334 Counter current low, 388 Creatine phosphokinase, 219 CRL, JAPAN, 359 Cross-linking reagent, cleavable, 104, 105 non-labeled, 103 stable isotope-labeled, 103 Cross-linking strategy, 102 afinity labeled, 103 solution cleavable, 103 stable isotope labeled, 103 Cross-linking, mixed isotope, MIX, 103 Cross-section, absolute, 216, 218 calibration standards of, 215 determination of, 214–216, 219, 221, 223, 227 rotationally-averaged, 208 Cross-sections, comparison of, 218, 219 normalized, 218

Crude oil analysis, 225 Crude vegetable extract, 439, 476, 477 Cryo electron microscopy, 221 Crystal formation, inhomogeneous, 432 Crystal-rich regions, 435, 436 CsI, 177, 178 cluster, 171 C-terminal residue, 19 C-trap, 14 Cu2+, 86 Cumene, 495, 498, 500 Cumene hydroperoxide, 500 Cyclotron frequency, 125, 132, 317 Cyclotron motion, 124, 293, 317 Cylindrical ion trap, CIT, 42, 328 Cysteine residue, biotinylation of, 86 Cytochrome c, 54, 141, 133 Cytochrome C peptides’ solution, 130

D D2, 311 D2O, 12, 36–55 D2S, 38, 43 Daidzein, 155, 156, 162, 163 Daidzein-7-O-glucoside, 155, 156, 162, 163 Damped harmonic oscillator, 303 Damping coeficient, 308 Danish Natural Research Foundation Centre for Quantum Optics, 324 Danish Natural Science Research Council, 324 Database search algorithm, 84 Data-dependent MS/MS method, 139 DC axial potential, 298 DC potential, 12, 341, 346–348 DC turning quadrupole, 419 DC voltage, 317, 339 DDS, see Scan, data-dependent de Broglie wavelength, 177 De novo sequencing, 84 DE50 value, 158, 159, 161–163 Debye–Scherrer rings, 176 Decabromodiphenyl ether, 482 Decaluorotriphenylphosphine, 467–469 Decomposition pathways, consecutive and competing, 373 Degrees of freedom, 157 Deinococcus radiodurans, 129 Deinococcus radiodurans proteome, 140 Dendrimer, 19 Density functional calculation, 182, 248, 251 Density functional theory, 179 Dentate gyrus, 425 Deoxyribose monophosphate nucleotide, 54 Derivatization strategy, 100 DESI, see Ionization, desorption electrospray Desorption ESI, DESI, 224

518 Detection eficiency, 320 Detection limit(s), 447, 491, 495–497, 505 Detector, 389, 394 Deuterating agent, 43, 45 gas-phase acidity and basicity of, 38 Deuteron transfer, 39 DFTPP, see Decaluorotriphenylphosphine DI, 38, 51 Diagnostic ion, 22, 87 Dialysis-related amyloidosis, 220 Dibromochloromethane, 495 Dichloroacetate, DCA, 432 Dichlorobenzene isomers, 498 Dichlorobromomethane, 495 Dichloroethane, 502 Dichloromethane, DCM, 408, 473, 475, 476 Diesel/oil extract, 473–476 Diethylpyrocarbonate, 101 Difenoconazole, 446 Differential isotopic enrichment, 98 Differential stable isotope labeling strategy, 102 Diffraction data, 177, 179, 180, 182, 184, 185 Diffraction pattern, 176, 177, 182–185 analysis, 177 calculated, 171 measured, 171 Digital ion trap, 367, 374, 275 Dimethyl methylphosphonate, 403, 406 Dipolar mode, 368 Direct current (DC) pulse, 370 activation, 369 Discharge source, 4 Disease studies, 425 Dispersed emission spectrum, 255–257 Dispersed luorescence, 255 Displacement reaction, 411 Dissociation yield, 241 Disulide bond, selective cleavage, 70, 137 Disulide linkage, 21, 22 DIT, see Digital ion trap DMDNP, see 2,3-Dimethyl-2,4-dinitropentane DMMP, see Dimethyl methylphosphonate DMNB, see 2,3-Dimethyl-2,3-dinitrobutane DMP, see 2,4-Dimethylpyridine DNA, 60, 246, 424 DNB, see 1,4-Dintitrobenzene Domain, frequency, 459 Domain, time, 459 Dominant conformers, interconversion among, 195 Doppler cooling force, 303, 312, 316 Doppler effect, 301, 312, 332, 339, 348 Doppler laser cooled, 299, 312, 319 Doppler proile, 333, 344 Doppler spectrum, calculated, 333, 334 DPM, see 3-(3-Methoxypropoxy) propanol Dried droplet method, 433

Subject Index Drift cell, 205, 207, 208 IMS, DCIMS, 206, 207 ion mobility–mass spectrometry, DCIM-MS, 207, 208, 215, 216, 218, 223, 224 resolving power, 225 Drift ield, electrostatic, 389 Drift gas inlet, 390 methane, 403 nitrogen, 403 Drift length, 397 Drift region, 387, 389, 390, 393 Drift time, 216, 390 Drift tube, 391 cylindrical, 397 DriftScope program, 213 output of, 214 Drosophila melanogaster, 208 Drosophila Toll receptor, 221 Drug, active, 435 Drug, Pro-, 435 Drugs, 419, 433, 435, 436 Duty cycle, 128, 378 fast, 67 rectangular waveform, 374, 375, 378, 385 Dye luorescence, quenching, 195 Dye–ligand afinity chromatography, 86 Dye–Trp proximity, 191 Dynamic peak range, 455

E e− - trapped ion interaction, 175 e− -beam-cloud overlap, 174 E/N, 391, 407, 408 ECD FT-ICR mass spectrum, 136, 137 ECD mass spectrum, 136 ECD of protein and peptide, 135–138 ECD, principles, 135 ECD, see Electron capture detection Effective collisions, 383 Effusive oven, 299, 342 EI, see Ionization, electron Electric circuit, switchable, 442, 443 Electric ield, 205 gradient, 388 strength, 390, 404, 408 dipole, 442 higher-order multipole, 442 quadrupole, 442 Electrical breakdown, 391 Electrodes, coated, 439, 444 hyperbolic angle of, 263 Electrodynamic ion trap, 3, 4, 13, 14, 16, 25 Electromagnetic trap, linear, 357 Electron afinity, 24, 406 Electron association reactions, 411

519

Subject Index Electron attachment rate constant, 407 Electron beam, pulsed, 439, 445 Electron capture, 387, 406 cross-section, 135 detection, 484 dissociation, ECD, 21, 64, 89, 123, 130, 246 dissociative, 400 rate constant, 407, 408 thermal, 387 Electron detachment reactions, 411 Electron detachment, thermal, 387, 406, 409 Electron diffraction, 170, 171 instrument, 172 measurement, 175 pattern, 175 Electron energy, 447 distribution, 407 Electron gun, 172, 297 Electron impact ionization, EI, 36, 342, 503 Electron multiplier, 173, 452 Electron multiplying charge-coupled device, EM-CCD, 257 Electron photodetachment, 241, 254 Electron scattering, inelastic, 176 total, 175 Electron transfer, ET, 12, 20, 24, 60, coupled with PTR, 67 ield-induced, 195 photo-induced, 191 plus CID, EtcaD, 64 Electron transfer dissociation, ETD, 6, 8, 15, 17–19, 21, 22, 24, 60, 64, 66, 67, 70, 72, 142 comparison with ETcaD, 65 non-dissociative, ETnoD, 64 multiple, 66 reaction, bio-ion/ion, 21 without dissociation, ET, 24 Electronic action spectroscopy, 252 spectrum, 252 Electronic excitation, 176 Electro-optic modulator, EOM, 302 Electro-optical chopper, EOC, 300, 302 Electrospray ionization, 3, 4, 7–12, 14, 15, 17, 24, 25, 36, 46, 47, 49, 52, 54, 62, 84, 91–93, 103, 154, 160, 207, 210, 215, 221, 253 source, 170, 257 orthogonal, 157 Electrostatic ield, 140, 194–196, 392, 403 interaction, 194 lens, 128 e-mail, tranmission of results from unmanned site by, 503 EM-CCD, Newton, Andor Technologies, 257 End lens, 70, 126–128

End-cap electrode(s), 7, 157, 173, 190, 191, 262, 269–273, 276, 303, 337, 341, 345, 347, 350, 367, 368, 372 End-cap electrodes trap, 345, 346 AC voltage applied across, 300, 301, 303, 434 stretched out, 442 Endoproteinase Lys-C digestion, 24 Endrin, 469 Energy-resolved mass spectrometry, ERMS, 155, 157–159, 161, 164 Energy-variation study, 54 Enthalpy, 388, 394, 396 changes, standard, 397, 399 reaction, 398 Entropy, 388, 394, 396 changes, standard, 397, 399 reaction, 398 Enzymic activity, 430 Epicatechin (−), 155, 156, 162–164 Equilibrium, 393 Equilibrium constant, 397 Equilibrium, phase-partitioning, 493 Equine myoglobin, 218 Escherichia coli, 16, 140, 144, 220, 241 ESI, see Electrospray ionization ESI-FT-ICR, 140 ESI-LIT-TOF instrument, 51 ESI-QIT instrument, 51 ETD, see Electron transfer dissociation ETD/CID MS/MS, 64 Ethylbenzene, 492, 495, 497, 498, 500 Ethylbromide, 402, 403 ETnoD, multiple, 66 European Union Directive 98/83, 492 Exact hard-sphere scattering, EHSS, 222 Excimer laser, 252 Excitation probability, 360 non-resonant, 367, 369 resonant, 367, 369, 373, 374 Exciton Corporation, 255 Exogenous compounds, 433 Explosives, detection of, 388, 399 External calibration, 14 Extract(s), contaminated, analysis of, 439 Extractive electrospray ionization, EESI, 224

F Faraday cup, 172, 173, 207 Faraday plate, 390, 398 Fatty acid tail(s), 426, 430 Fatty acyl chains, 423 FC-43, see Perluorotributylamine Fe3+, 86 FeCO2−, 70

520 FELIX, 246, 248, 249 Fiber optic, 420 Fick’s equations, 493 Field adjusting phase, 375–377 Field asymmetric waveform IMS, FAIMS, 206, 209, 228 Field test(s), 491, 496, 502, 505 Filament assembly, 448 Filtered noise ield, 459, 460 Finite-element based program, 345 Finnigan 3D QIT, 67 Corporation, 466 LTQ, 420, 423 LTQ mass spectrometer, 9, 69, 71 Finnigan MAT, 440 First-Doppler effect, 332, 343 First-order Doppler shift, 342, 358 Fixed-charge derivatization, 91 Flavonoid, 154–157, 162–164 Flavonoid glycoside isomers, 49, 50 Fluoranthene, 9, 476 Fluoranthene anion, 68–70 Fluorescence, 294, 300, 334, 341, 348, 353, 418 decay, 190 detection sensitivity, 188 emission, 242, 256, 342, 349, 350, 353 spectrum, 186 excitation spectrum, 255, 256 hole, 266, 270, 271, 274, 275, 278 image from 40Ca+, 300, 307, 308 imaging system, 300 intensity, 187–189, 281, 348 lifetime, 186 measurement, 191, 193 lifetime, temperature dependence, 193, 195, 198 measurement, 191, 334 resonance energy transfer, FRET, 255, 283 spectroscopy, 240, 242, 254, 255 Fluorescent lifetime, 191, 192 Fluorophore, 418 FNF, see Filtered noise ield Focusing device, 128 Forbidden optical transition, 332 Forward mass scan, 376, 377 Fourier transform, 174, 191, 460 ion cyclotron resonance, FT-ICR, 9, 15, 19, 42, 44, 45, 51, 121–144, 207, 242, 244–246, 255, 282, 293, 95 analysis, 126 instrument, 51 mass spectrometry, 388, 389, 458 principles of, 122–126 resolving power, 129 sensitivity, 130

Subject Index Fragmentation reaction, selective gas-phase, 107 threshold, 465 site-speciic, 93 Franck–Condon factor, 24 Free energy, 388 Free-electron laser, FEL, 244, 246, 247, 251, 282 Free-jet expansion, 396 Frequency metrology, 328, 341, 357–359 Frequency of ion motion, 5 Frequency power spectrum, 269, 270 Frequency reference, 354 Frequency spectrum, 126, 348, 349, 460, 462 Frequency stability, 358 Frequency standard, 358 Frequency synthesizer, programmable, 301, 302, 305 Frictional force, 312, 313 FT-ICR, see Fourier transform ion cyclotron resonance Fundamental secular frequency, ωr,0, 262, 263, 268, 298, 337, 339, 343, 349, 350 Fundamental secular frequency, ωz,0, 262, 263, 268, 271, 298, 337, 339, 343, 349, 350 Fungicide, 446

G Ga3+, 86 Gas chromatography/mass spectrometry, GC/MS, 49, 439, 476, 478, 486, 496 Gas chromatography/tandem mass spectrometry, GC/MS/MS, 439, 440, 442, 454, 455, 457, 470, 486 Gas-phase acidity, 39, 40, 49 basicity, 39, 40 ion, 3 ion chemistry, 41 ions of opposite polarities, 3 reactions, 394 Gaussian peak, near-, 392 Gelatin, 154 General relativity theory, 330 Geometric parameter, η, 298 German National Institute, 345 Glucokinase, 133 Glucose polymer, 227 Glutamine, 129 Glycerol backbone, 423, 425 Glycerophosphocholine lipids, 22 Glycopeptides, 227 Glycoprotein, 134 Glycosylation, 21 Gravitational redshift, 359 Gravity wave, 330

521

Subject Index Greenwich Mean Time, GMT, 329 Grid(s), 389 Ground-positioning system, GPS, 330, 354

H H/D exchange analysis, 53 mass spectra, 48, 49 reaction, 37, 39 historical perspective, 36 deuterating agents, 38 doubly-protonated species, 47 lip-lop mechanism, 40 instrumentation, 42 ion trapping, 42 model compounds, 47 model peptides, 51 motivation for, 41 practical aspects, 40 proposed mechanisms, 38 protein, 51, 53 theory of, 37 H2, 310, 311 Halobacterium salinarum, 142 Haloethanes, 492, 497 Halomethanes, 492, 497 Hard-sphere model, 207, 264, 273 Harmonic potential, one-dimensional, 302 HASTE CID, see Collision-induced dissociation techniques, HASTE CID Hazardous substances, monitoring of, 388 HCT, see High Capacity Trap HD, 310, 311 Head-space, 500 Heart disease, molecular differentiation, 140 Heavy gases, presence of, 372 Heidelberg, 345 Helium, 368, 372, 375, 383, 440, 447 Heme, 430, 432 Hemoglobin, 219 Hemoglobin (Hb) tetramer, 215 Hepatitis B virus capsid protein, 220 Hepatitis C patient, cryoglobulins, 140 Heptabromodiphenylether, 480 Heptachlor epoxide, 469 Hewlett-Packard-Austin, 353 Hexapole ion trap, 340 Hexapole LIT, 14 Hg+, 346, 352–355, 357 High amplitude low frequency, HALF, 16 High amplitude short time excitation, HASTE, 98 High capacity trap, Bruker, 444 High performance liquid chromatography, HPLC, 154, 223, 435 /tandem mass spectrometry, 436 High resolution mass analysis, 14

High sensitivity, 15 High-energy synchrotron radiolysis, 101 Higher-order ield, 262–264, 268, 271, 315, 316 Higher-order terms, 314, 315 HighQ Pulsed CID, see Collision-induced dissociation techniques, HighQ Pulsed CID Histidine, 24, 127 Histone PTM state, 70 Hitachi 3DQ mass spectrometer, 42 Hitachi M-8000 ion trap mass spectrometer, 6 Hole, in electrode, 260, 262–266, 269, 270–275, 279, 334 Homochirality, 228 Hot electron capture dissociation, HECD, 137, 140 HPLC, see High performance liquid chromatography HPLC/MS/MS, see High performance liquid chromatography/tandem mass spectrometry Human cerebrospinal luid, 140 Hb variants, identiication of, 228 HeLa cell, 144 nuclear protein, tryptic digest, 21 serum, 85 α-casein, 143 Hybrid FT-ICR instrument, 123, 131, 132, 138, 140 Hybrid instruments, 15 linear ion trap FT-ICR, 138, 139 LIT/FT-ICR instrument, 14 Q-TOF instrument, 213, 214 tandem mass spectrometers, 13 LIT /FT-ICR, 13 Orbitrap, 13 quadrupole/TOF, 13 triple quadrupole/LIT, 9 Hydrate ions, 396 Hydration reactions, 397 Hydrogen atom, labile, 45, 48, 49 non-labile, 49 Hydrogen bonding, intramolecular, 48 Hydrogen/deuterium (H/D) exchange, 36–55, 101, 221, 223 reaction, 36 Hydronium ion, 395 Hydroxyl radical probe, 101 Hyperbolic rods, 338 Hyperboloidal ion trap, 334, 345 Hyperine transition, 332

I I−, 70 IA, 64, 71 IA coupled with CID, 70

522 Icosahedral capsid, 220 ICR cell, 122–144, 242, 243, 246 Image creation, 417, 421 Image current, 126 Images, chemically-selective, 418 Imaging, 225 mass spectrometry, 417, 426, 430, 435 spatial resolution, 225 system, 319, 320 IM-mass spectrometry, reviews, 209 Immunoafinity chromatography, 86 IMS, see Imaging mass spectrometry IMS, see Ion mobility spectrometry IMS/MS, see Ion mobility spectrometry/mass spectrometry IMS-Q-TOF mass spectrometer, 208 In+, 355 In vitro chemical derivatization, 94 In vivo metabolic labeling, 94 Incident angle, 420 Informing power, 15 Infrared chromogenic cross-linker, IRCX, 109 Infrared multi-photon dissociation, IRMPD, 41, 109, 123, 133, 243, 244–247, 251–253, 282 In-source collision-induced dissociation, ISCID, 105 Instrument duty cycle, 12 Intermediate electrode, 374 Internal atomic oscillator, 330 Internal calibration, 14 Internal enegy, 369, 465 deposition, 368, 369 Internal standard, 433, 434 International Atomic Time, TAI, 329 Intersystem crossing rate, 198 Intramolecular interaction energy, 197 Intramolecular vibrational redistribution, IVR, 245, 249–251, 253 Ion activation, 5, 370, 439, 461 data-dependent, 64 infrared photon, 64 Ion attachment, IA, 60 Ion charge control (ICC) value, 281, 282 Ion clocks, current research, 352 Ion cloud, 174, 175, 188, 242, 243, 255, 258, 260, 264, 279, 280, 335, 337 overlap, 10, 264, 280, 348 size estimation, 278, 345 imaging, 342 increased density, 189 linear, 334 manipulation, 170, 342 spatial distribution, 278, 335, 348, 350, 358 trapped, 173, 349, 351

Subject Index Ion cyclotron motion, 123 Ion cyclotron resonance cell, 124, 242 mass spectrometer, 37 Ion detection, 11 eficiency, summary, 274 Ion ejection, 272 eficiency, summary, 274 Ion ensemble, spatial distribution of, 279 Ion luorescence, 170, 307, 309, 310 Ion fragmentation, 7, 11, 212 Ion genealogy, 5 Ion guide, 13 RF-only, 208 Ion injection, 7, 10, 11 eficiency, 11 Ion internal energy, 61, 158 Ion isolation, 5, 10, 439, 455 Ion kinetic energy, 12, 369, 372, 446, 447 Ion manipulation, 72 Ion micro-motion, 345, 353, 359 Ion mobility spectra, 391 Type 1, 393, 395 Type 2, 393, 400 Type 3, 394, 403 Ion mobility spectrometer, hand-held, 396 Ion mobility spectrometry, IMS, 205, 387, 388, 417, 419 Ion mobility spectrometry/mass spectrometry, 394, 397, 399, 402, 403, 411 Ion mobility, IM, 205, 207 Ion mobility-mass spectrometry, 205–230 traveling wave, 205–230 applications, 205–230 Ion motion detection, 302 Ion motion frequency(ies), 348 Ion motion in QIT, dynamics of, 261 Ion motion, theoretical treatment, 262 Ion number density, 12, 16 Ion optical clock, 355 Ion parking, 16, 17, 25, 66 Ion photo-excitation, 242 Ion processing, multi-stage, 63 Ion production region, 310 Ion reaction vessel, 60 Ion selection, 370 Ion shutter, 389, 390, 392 Ion source, external, 446 moveable, 397 multiple, 62 Ion spin exchange, 328 Ion splat events, axial distributions of, 273 Ion storage time, variation of, 36 Ion swarm, 390 Ion temperature, 316, 319, 343, 354 Ion tomography study, 278

Subject Index Ion trajectory calculation, 263–266 Ion trajectory simulation, 189 Ion trajectory sImulation software package, ITSIM, 264, 276 Ion trajectory, Fourier analysis of, 263, 266, 268, 271 stable, 261, 262, 268 unstable, 262 Ion transmission mode, 12 Ion transmission time, 12 Ion transmission, eficiency, 7 Ion trap detector, 440 Ion trap dimensions, 5 Ion trap geometry(ies), 328 Ion trap housing, 258 Ion trap imperfection, 315 Ion trap loading, 317, 319, 321 Ion trap mass spectrometer, 60, 170, 187, 190, 239–284, 492 Ion trap parameters, choice of, 323 Ion trap technology, 170 Ion trap, 2D LC/MS, 447 Ion trap, micro, 295 Ion trap, miniature, design, 343 Ion trap, non-linear, 439 Ion trap, Paul-Straubel type, 347, 349 Ion trap, quadrupole, 455 Ion trap, quasi-miniature, 339 Ion trap, segmented, 338 Ion trapped simultaneously, 299, 307, 323 Ion trapping, 328 charge-sign independent, 63, 64 eficiency, 11, 349, 446 parameter, 261, 297, 320 technique, 328 Ion traps, millimeter-scale, 345, 352, 357 multi-pole, 335, 336, 339 Ion, doubly-charged, 223 forced motion of, 300-303 metal cluster, 169, 170, 173 multiply-charged, 241 sequence-informative, 70 Ion/ion chemical reaction, 60 Ion/ion chemistry, 7, 12, 15, 61, 72 Ion/ion ETD reaction, 9, 14 Ion/ion interaction, 348 Ion/ion proton transfer reactions, 15 Ion/ion reaction, 3, 4, 6, 8–11, 15, 17–20, 25, 61–63, 66, 68 cation-switching, 20 eficiency, 5 kinetics, charge-squared dependence of, 16 sequential, 14 tools, 4 vessel, 14, 64 Ion/molecule chemistry, 389

523 Ion/molecule interaction potential, 396 Ion/molecule processes, 392 Ion/molecule reaction, 62, 207, 387, 393, 395, 396, 445–467 association, 411 time, 446 Ion/molecule research, 440 Ion/neutral association, 392 Ionization eficiency, 12 Ionization energy, 447 Ionization time, 372 duration of, 441, 466 ixed, 466 Ionization, ambient, 225 Ionization, chemical, 36–40, 63, 69, 439, 447, 448, 453, 466 external, 450 hybrid, 448, 451, 454, 486 internal, 451 liquid, 439, 448–450, 481, 486 negative, 451 positive, 451 pulsed positive ion negative ion, see PPINICI reagent, 448, 481 gas pressure, 448 ions, 449 selective-ejection, 448, 449 self-ejection, 448 source, 70, 71 /tandem mass spectrometry, 483, 484 Ionization, desorption electrospray, 418 Ionization, electron, 299, 310, 444, 447, 453, 466 Ionization, internal, 444, 445 Ionization, self-chemical, 445 Ion-manipulation methodology, 60, 64 Ion–molecule complex, 37, 40, 42, 44 Ions, collisional cooling of, 262 Ions, laser-cooled, 345 Ions, mobility separation of, 212 Ions, negative, 388, 393, 406, 411 Ions, positive, 388, 411 Ions, precursor, 456, 457 Ions, simultaneous trapping of both polarities, 9 Ions, spectroscopy of, 239–284 Ion-trap dimension, r 0, 298 z0, 298 Ion-trap electrodes, 318 Ion-trap frequency, 320, 321, 323 relative shift, 318 Ion-trap-oscillator ensemble, 328 Ion-trapping device, 227 gas-phase, 73 IP-MALDI, see Matrix-assisted laser desorption, intermediate pressure

524 IRMPD action spectrum, 247–249 IRMPD FT-ICR mass spectrum, 134 IRMPD MS/MS, 140 IRMPD of protein and peptide, 133–135 IRMPD, principles, 133 Iron-containing ions, 6 ISCID mass spectral scan, 106 Isoaspartic acid residue, 22 Isobaric interferences, 429, 432 Isobaric ion identiication, 417, 427 Isobaric ions, 428, 436 Isobaric separation, 429 Isobaric species, 427 Isobars, 427, 428 Isolation resolution, 457 Isolation window, 428, 433, 434 Isolation, low mass, 456 Isolation, notch, 457, 460 Isolation, two-step, 456, 457 Isoleucine, 137, 140 Isomer differentiation, 47, 49 Isomer diffraction pattern, calculated, 183 Isomer space illing structure, 181 Isophthalic acid, 48 Isotope effect, 311 Isotope-coded afinity tag, ICAT, 94, 96, 97 Isotopic cluster ions, 478 Isotopic patterns, 503 Isotopic peaks, 441 Isotopomer peak, 458 Isotopomers, 378 Italy, laws, 492 ITD, Finnigan Corporation, 370 ITD, see Ion trap detector ITD-700, 466 ITD-800, 466 ITMS, see Ion trap mass spectrometry ITQ, see Thermo Scientiic ITQ, 451 iTRAQ approach, 97, 98 iTRAQ reporter ion, 99 iTRAQ-labeled peptide, 100 ITS-40, 440, 467 IVR killer mode, 251

J Jet Propulsion Laboratory, JPL, 329, 337, 339, 340, 352, 353, 355 JPL geodetic receiver, 354 Jumping, 377

K Kinetic constants, 394 Kinetic data, 387, 391, 394 Kinetic energy, 382, 383, 391, 431, 464

Subject Index Kinetic shift, 243, 245, 253 KinFit, 45, 47 Knudsen oven, 178

L Laboratoire national de métrologie et d’essais, Système de références temps espace, LNE-SYRTE, 331, 353 Lamb-Dicke parameter, 333, 339 regime, 333, 335 Laser beam, 242, 295, 297, 299, 323, 334, 347, 419 Laser capture microdissociation, 435 Laser cooling, 294, 301–304, 311–313, 318–321, 328, 332, 333, 335, 339, 341, 342, 356, 357 process, 358 Laser desorption/chemical ionization, 419 Laser diode, 328 Laser dye, 252 Laser hole, 266, 270, 271, 274, 275, 278, 280, 339 Laser modulation, 303, 305 Laser power, 281, 282, 422, 423 Laser spot size, 421 Laser tuning, 360 Laser, CO2, 243-246 Laser, ixed-wavelength, 240, 242, 243 Laser, Nd:YAG, 243, 252, 257 Laser, nitrogen, 243 Laser, titanium:sapphire, 252, 257, 281, 357 Laser, tunable IR, 244, 245, 247, 256 Laser, vacuum UV luorine, 244 Laser-cooled mercury ions, string of, 339 Laser-induced luorescence, LIF, 255, 257 Laser-induced reaction spectroscopy, 250 LC CID MS/MS analysis, 141 LC/MS, see Liquid chromatography/mass spectrometry LC/MS/MS, see liquid chromatography/tandem mass spectrometry LC/MSn, see Liquid chromatography/tandem mass spectrometry LC-ESI-MS, 156 LCM, see Laser capture microdissociation LC-MS/MS analysis, 85, 86, 96, 100, 140 LCQ, 444 Advantage ion trap mass spectrometer, 157 LD/CI, see Laser desorption/chemical ionization Leak valve method, 44 Lectin afinity chromatography, 86 Leucine, 137, 140 Leucine encephalin, protonated molecule of, 379–381

Subject Index Lifetime measurement, 193, 194 time-resolved, 189 Linear extended ion trap, LITE, 339, 354 Linear ion trap standard, LITS 1–4, 354 Linear ion trap standard, LITS project, 352 Linear ion trap, LIT, 9–11, 15, 24, 25, 42, 62, 63, 69, 86, 92, 98–100, 255, 294–297, 300–302, 310, 311, 313, 334, 337, 359, 424, 425, 442 chamber, 52 quadrupole array, 9 sketch of, 296 Linear polarizer, 302 Linear trap ensemble, 340 Linear two-dimensional (2D) quadrupole ion trap, 9, 337, 339 Linear two-ion system, 293, 294, 300, 303, 305, 307, 308, 315 Linearity, 491, 498, 505 Lipids, 419, 422, 424 Liquid chromatography, LC, 84, 208 /mass spectrometry, LC-MS, 49, 422, 442 /mobility separation, 228 -tandem mass spectrometry, LC-MS/MS, 64, 422, 442 Liquid nitrogen, 175 LIT, see Linear ion trap LIT-CID MS/MS, 141, 142 LIT-FT-ICR instrument, 140, 143 LIT-TOF system, 53 LMCO, see Low-mass cut-off Local atomic oscillator, 330 LOD, see Detection limits Logic atomic clock, 356 Low-critical energy process, 371 Low-mass cut-off, LMCO, 5, 64, 101, 104, 241, 372, 439, 465 LTQ, 420, 431, 432, 442 Orbitrap XL, 14 Luteolin, 155, 156, 162, 163 Luteolin-4′-O-glucoside, 50, 155, 156 Luteolin-7-O-glucoside, 50, 155, 156 Lysine, 24, 127, 129 Lysophosphatidylcholine, 430 Lysozyme, 54, 141

M Machinable macor, 346 Macromolecule ions, 19 metal-containing, 21 Macromolecule mixture analysis, 15 Macro-motion, 348, 352, 353 Magnesium, 319 Magnetic ield, Earth, 317 residual, 317 Magnetron motion, 124

525 MALDI, 15, 42, 46, 84, 93, 126, 139, 156, 207, 210, 215, 225, 241, 243, 417–419, 422, 424, 429, 430, 433, 435, 436 MALDI-FT-ICR, 140 MALDI-QIT instrument, 51 Malondialdehyde, 88 Mapping, 377 Marseille, 329, 347, 359 Mass accuracy, 14, 15, 468 Mass analysis, 11, 13 data-dependent, 65 high-sensitivity, 67 Mass analyzer, 14 Mass discrimination, 392 Mass ilter, 13 Mass isolation window, 10 Th, 157, 159 Mass range, 419 Mass ratio μ, 296, 303, 314, 322 Mass resolution, 310, 324, 468 higher, 439, 442 Mass spectrometric imaging, full-scan, 417, 429 tandem, 328, 417, 429 Mass spectrometry region, 310 Mass spectrometry, identifying ions by, 392 Mass spectrometry/mass spectrometry, see Tandem mass spectrometry Mass spectrum, ‘quadrupole like’, 468 Mass spectrum, simulated, 274–277 Mass spectrum, Synapt, 214, 219 Mass-resolving power, 14, 15 Mass-selected ions, optical spectroscopy of, 240 Mass-selective axial ejection, MSAE, 9, 11 instability scan, 371, 375 Mass-selective external ion accumulation, 138 Mass-selective instability, 259, 264, 272, 273, 276, 440, 441, 456 Mathieu equation, 348, 374 Mathieu parameters, 374 Matlab v. 7.0, The Mathworks, Inc., 228, 266 Matrix cluster ions, 429 Matrix effect(s), 12, 491, 499 Matrix ions, 424 Matrix solution, 421 Matrix-assisted laser desorption, atmospheric pressure, AP-MALDI, 419 intermediate pressure, 417, 430 intermediate vacuum, 420, 423, 431, 432 ionization, see MALDI Max Planck Institute, 359 m-Diluorobenzene, 37 Medial genticulate body, 425 Melittin, 67, 68 Membrane inlet mass spectrometry, MIMS, 491, 493, 494, 500–502, 505 Mercury, 327, 332, 334, 352, 356 Mercury ion, 329, 332, 334, 337, 339, 352 Messenger spectroscopy, 250

526 Metabolites, 418, 419, 422, 424, 435, 436 Metal cluster aggregation sources, 170–172 collisional relaxation, 175 ion source, 174, 175 Metal transfer, 20 Metal-ion afinity chromatography, IMAC, 86 Metal-ion insertion, 25 Metal-ion transfer, 15, 19, 20 gas phase, 20 Metal–oxide afinity chromatography, MOAC, 86 Metastable state, 342 Methane, 403 Methanococcus jannaschii, 144 Methanol, 481 Methionine residue, 91 Method detection limit, MDL, 485, 486 Methyl bromide, 401–403, 411 Mg+, 295, 299, 311 MgD+, 305, 310, 311 MgH+, 305, 310, 311, 324 Microcrystals, 435 Micro-extraction, 484 Micromotion, 316, 344 amplitude, 334, 335 minimization of, 344 Microwave frequency domain, 329, 333, 352 Microwave frequency standard, 334 Microwave interrogation, 339 Microwave-assisted D-cleavages, 24 Military preparedness, 411 Minimum energy conformation, 248, 251 Mirror image, 256 Mobile proton condition, 87, 90 Mobilities, differences in, 390 Mobilitiy measurement(s), 387, 389 Mobility cell, 227 Mobility coeficient, 396 Mobility data, 213, 226 Mobility gas, 218 Mobility separation, 224, 228 Mobility spectrum, 387 Mobilogram, 213, 214 Model proteins, top-down study, 18 Modes, electronic, 388 rotational, 388 translational, 388 vibrational, 388 Modiication, S-type, 108 unique, 107 Modulation frequency, 305 Modulation voltage, 300, 301, 307 Molar Gibbs energy, ΔG, 38 Molecular beam, 295 Molecular dynamics, 161, 191, 219, 251 simulation, 191, 195, 197, 207 Molecular ion cluster, 379

Subject Index Molecular ion, photodissociation, 311, 323 Molecular ions, 310, 323 Molecular mechanics force ield, 251 Molecular scattering curve, 178, 179 Molecular scattering data, 180 Molecular scattering intensity, 177, 178, 181, 182 Molecular weight distribution, 227 Molybdenum, 337, 345, 347 Monitoring, 388 prolonged, 491 Monoclonal antibodies, glycosylation of, 226 Monoisotopic mass, 129 Monoisotopomer, 378, 379 MS/MS, see Tandem mass spectrometry MSn, see Tandem mass spectrometry, multiple stages of Multiphoton dissociation, 250, 251 Multiple collisions, 369 Multiple decomposition channels, 369 Multiple photon process, 250 Multiple resonant frequencies, 480 Multiple stages of mass selectivity, MSn, 60 Multiply-charged anions, 7 Multiply-charged cations, 6 Multiply-charged ion, 3, 4, 18, 19, 61, 127 Multiply-charged precursor ions, 17 Multiply-charged reagent ions, 19 Multiply-protonated polypeptides, 6 Multipole storage-assisted dissociation, MSAD, 131 Multipoles, higher-order, 443 Multi-sector mass spectrometers, 368 Mutual ion storage, 6, 10 Mutual ion storage mode, 9, 13, 14 Mycobacterium tuberculosis, 144 Myoglobin, 133, 215, 218

N NADH dehydrogenase 1 beta sub-complex 3, 99 n-Alkyl bromide, 400–402 Nanoelectrospray ionization (nESI) source, 187, 190 Nano-liquid chromatography, 130 Nano-spray static tip, 62 Naringenin, 155, 156, 160, 162, 163 Naringenin-7-O-neohesperidoside, 155, 156, 160, 162, 163 NASA Deep Space network, 354 National Burean of Standards, NBS, 329 National Institute of Standards and Technology, NIST, 251, 329, 337, 338, 346, 353, 354, 356, 357, 396, 398 National Institutes of Health, 55 National Physical Laboratory, NPL, 329, 346, 359 National Research Council, NRC, Canada, 359

527

Subject Index Natural Sciences and Engineering Research Council, Canada, 284 N-Benzyliminodiacetoylhydroxysuccinimid, BID, 104 NCI, see Ionization, chemical, negative ND3, 37, 38, 40, 43, 51, 54 NDBA, 483 NDEA, 483 NDMA, 483 NDPA, 483 Negative chemical ionization source, 15 Negative ETD reagent ions, 14 Negative ions, 15 Neohesperidin, deprotonated, 47 Nephelometry, 154 Neurotensin, cross-linked, pELYENKPRRPYIL, 107, 108 Neutral loss, NL, 429 Newton’s equations of motion, 264 NH3, 329 Nickel acetate, 20 NIST Chemistry WebBook, 38 Nitrobenzene, 6 Nitro-compounds, 472 Nitrogen, 403, 406 Nitrophenols, 472 Nitrosamines, 483 NMEA, 483 Nobel Prize for Chemistry (2002), 127 Nobel Prize for Physics (1989), 328 Nomenclature, backbone product ions, 131 Non-exchanged isotopic contribution, 45 Non-linear ield component, 260, 261, 263 Non-linear ion trap, see Ion trap, non-linear Non-linear resonance, 263, 268, 348, 350 Non-mobile proton condition, 87 Non-zwitterion, 247–249 Normalized collision energy, NCE, 463 Notch window, 460 Nozzle-skimmer dissociation, 131 NPIP, 483 NPYR, 483 N-terminal derivatization, 88 Nuclear magnetic resonance, NMR, 221 spectrometry, 154, 228 Nucleic acid, 54 mixture analysis, 16 Nucleoside, 54 Numerical simulation, 298

O O2, 319 Oak Ridge National Laboratory, 492 Octopole ion guide, 9, 15 o-Diluorobenzene, 37 Ofice of Water, 468

Off-resonance excitation, 41, 132 Oleic acid, 426 Oligodeoxynucleotide anions, 20 multiply-charged, 20 Oligonucleotide, 15, 54, 223 One-liter trap, 340, 341 Oppositely-charged ion populations, 4 Optical cavity-laser, 355 Optical frequency comb technique, 356, 357 Optical frequency domain, 333, 341 Optical frequency ion clock, 358 Optical frequency standards, clocks for, 341 Optical parametric oscillator, OPO, 247, 252 Optical spectrum, 240 Orbitrap, 12 Orbitrap mass spectrometer, 100 Organohalogen compounds, 497, 503, 505 Organomercurial agarose bead, 86 Organophosphate chemical warfare simulant, 228 Organophosphorus compounds, 403 Orthogonal relectron TOF mass analyzer, 13 Oscillation frequency, 299, 303, 305, 308, 309, 314–316, 318, 319 Oversampling, 422

P P&T/GC/MS, see Purge-and-trap/gas chromatography/mass spectrometry Paclitaxel, 419 Pair correlation function, Fourier transform of, 171 Parallel ion parking, 16, 17, 25 Partial proton transfer reactions, 17 Paul trap, 5, 341, 349, 351; see also Quadrupole ion trap ion trap, 240, 242, 245, 246, 255, 257, 296, 346 PBDE-100, see 2,2′,4,4′, 6-Pentabromodiphenylether PBDEs, see Polybrominated diphenyl ethers PC, see Phosphatidylcholine PCBs, see Polychlorinated biphenyls PCI, see Ionization, chemical, positive PD, see Photodissociation PE, see Phosphatidylethanolamine Peak tailing, chromatographic, 445 Penning trap, 245, 296 Pentachlorophenol, 472 Pentapeptide, protonated, 51 Peptide backbone bond, multiple breakage, 67 Peptide backbone, 19 Peptide cation, multiply-charged, 71 Peptide identiication, 96

528 Peptide ions, 372, 382 differentially-labeled, 97 multiply-protonated, 24 Peptide mass ingerprinting, 139, 140 Peptide mixture, simple, 19 labeled, 97 Peptide sequencing, 140 Peptide subset, chemical derivatization of, 86 Peptide, amino acid sequence, 84 analysis, 121 cross-linked, 102, 106 cysteine-containing, 86, 92, 101 dead-end modiied, Type 0, 102 doubly-protonated, 71 highly-charged precursor, 67 histidine-containing, 86 identiication of cross-linked, 105 intermolecular cross-linked, Type 2, 102–105, 107 intramolecular backbone bonds, 70 intramolecular cross-linked, Type 1, 102, 104 methionine-containing, 91 methionine sulfoxide-containing, 87 N-acetylgalactosamine-containing, 87 phosphorylated, 86, 223 photodissociation, 109 Peptide–polyphenol, 153–164 gas-phase afinity scale, 155, 161, 164 supramolecular assembly, 153–164 tannin, 153 Peptides, 419, 422, 424 multiply-deprotonated, 21 three-dimensional structures of, 207 Perluoro-1,3-dimethyl-cyclohexane, PDCH, 6, 67 Perluorotributylamine, PFTBA, 452, 454, 457 Periaueductual gray, 425 Period, injection, 454 isolation, 454 Permanent magnet, 126 Permittivity of vacuum, 302, 337 Pervaporation, 493 Pe-scan, 444 Pesticide(s), 477 Phase method, 320, 321, 323 Phase shift, 272 Phase-detection method, 305, 309 Phenanthrene, 452, 453, 455 Phenylboronic acid, 101 Phenylisothiocyanate, 89 Phosphatidylcholine, 423, 425–428, 431 Phosphatidylethanolamine, 428–430 Phosphatidylinositols, PI, 424 Phosphatidylserine, 428–430 Phosphocholine, 425 head group, 431

Subject Index Phospholipid ion, 429 Phospholipid(s), 422, 425, 426, 432, 433 Phosphopeptide ion, deprotonated, 19 Phosphopeptide ions, 19 CID, 22 doubly-protonated, 19 ETD, 22 methyl-esteriied, 21 Phosphoric acid, losses of, 21, 142, 143 Phosphorus hexaluoride, 70 Phosphorylation, 21, 137 Photodissociation, PD, 240, 241, 252, 257, 282, 283, 369 Photoelectron spectroscopy, 182 Photo-induced dissociation, PID, 240 Photoionization beam, UV, 318, 319, 342 Photoionization, resonance-enhanced, 299 Photon detection, 319 PHPMS, see Pulsed high pressure mass spectrometry Phthalates, 472, 473 Phthalic acid, 48 isomers, 48 Physikalisch-Technische Bundesanstalt, PTB, 345, 359, 360 Planck’s constant, 341 Plasma chromatography, 206, 388 Plasma ECD, 138 mass spectrum, 138 Polarized compounds, 444 Polarized light microscopy, 435 Pole number, 336 Polybrominated diphenyl ethers, 478, 480, 481 Polychlorinated biphenyls, 439, 483–485 Polycyclic aromatic hydrocarbons, 392 Polyethylene glycol, PEG, 227 Polynuclear aromatic compounds, PNAs, 452, 454, 473 Polypeptide, 15 cation, multiply-charged, 21 ion, 9, 20 multiply-protonated, 21 Polyphenol, structure of, 153, 155 Polyproline lifetime measurements, 191 Polyproline peptide, 186, 190, 191 dye-derivatized, 189, 191 Polyvinylpolypyrrolidone, PVPP, 154 Porapak, 498 Porcine elastase, 18 Position speciic mass spectrum, 421 Positive ion mode, 426 Post-ion/ion reaction (PTR) MS3 spectrum, 68 Post-mortem human brain tissue, 434 Post-translational modiication, see PTM Potabilization, 497, 502 Potential well depth, 195, 299, 319, 335, 336, 339, 342, 345, 347, 350

Subject Index PPINICI, 448, 451 PQD-MS/MS, 98–100 Precision mass measurement, 309, 315 Precursor ion, 367, 368, 372, 373, 419, 427, 464, 465, 472 charge-state manipulation, 15 doubly-charged, 383 isolation, 61, 131 Precursor ions, higher-charged, 67 Pre-exponential factor, 405, 406, 411 Pre-ion/ion reaction (PTR) MS/MS spectrum, 68 Pre-scan, 422 Pressure, effect of increasing, 430 Prion diseases, 220 Prion protein, 220 Product ion, 367, 368, 383, 385 diagnostic, 91 manipulation, 18 mass spectra at selected working points, 384 in silicio, 18 mass spectra, simpliication, 15, 17 mass spectrum, 10, 18, 21, 23, 66, 69, 84, 90, 91, 96, 108, 227, 372 multiply-charged, 69 selected ejection of, 373 transition, 427 a-type, 88, 89, 137 b-type, 67, 87–89, 98, 100, 109, 132, 133, 137 c-type, 69, 136, 137 y-type, 67, 87–89, 98, 100, 109, 132, 133, 137 z-type, 69, 136, 137 Product ions, 428 singly-charged, 383 Projet d’Horloge Atomique par Refroidissement d’Atomes en Orbite, PHARAO, 332 Proline residues, 16 Proline rich protein, PRP, 154, 155 primary structures, 156 synthetic, 154 synthetic B714, 155–157, 159, 161–164 IB8c, 155, 156, 163 IB934, 155, 156, 160, 162, 163 Protein A, 134 Protein analysis, 16 Protein and peptide depletion, 85 separation, 85 Protein backbone cleavage, 71 Protein conformation, 224 Protein database search, 21 Protein digestion, enzymatic, 59 Protein expression, 93 Protein folding, 101, 209 dynamics, 101 Protein identiication, 14, 85

529 Protein interaction reporter, PIR, 105 Protein ions, fragmentation of, 16 Protein mis-folding diseases, 220 Protein mixture, 17 analysis of, 15 Protein quantitation, 93, 95 analysis, label-free, 94 stable isotope label, 94 Protein sequence analysis, 61 Protein sequence characterization, 72 Protein Sprouty2, 143 Protein stability information, 220 Protein structural characterization, 83 Protein structure, 101 Synapt, 219 Protein topology, low-resolution structure of, 101 Protein, analysis, 121 conformations of, 53 differential quantitative analysis, 93 highly-charged precursor, 67 multiply-protonated, 52 multiply-deprotonated, 52 Protein–polyphenols, interaction of, 154 Protein-protein interaction, 101, 102, 105, 106 Proteins, 422, 424 lubricating salivary, 154, 164 Proteome, human plasma, 208 Proteome, mouse brain, 86 Proteome, urinary, 208 Proteomic analysis, amniotic luid, 140 Proteomics approach, LC-ESI-MS/MS, 229 Proteomics research, MS-based, 84, 223 Proteomics, application of FT-ICR, 139 Proteomics, bottom-up approach, 70, 84, 85, 101, 123, 139 Proteomics, major goals, 84 Proteomics, shotgun approach, 84, 140 Proteomics, top-down, 18, 60, 123, 139, 143 Proton afinity, 392, 449 Proton hydrate, 395 Proton mobility, 106 Proton transfer, 11, 12, 15, 24, 39, 64, 105, 395 multiple, 12, 19 reaction, PTR, 6, 16–18, 60, 449 sequential, 19 Proton-bound dimer(s), 403, 404, 406 Proxy marker, 60 PS, see Phosphatidylserine Pseudo-irst order, 392, 401 Pseudo-potential trapping well, 5, 6, 339 Pseudopotential well, cross-section, 335, 336 PTM, 14, 59, 64, 72, 136, 142 PTM information, 21, 26 PTM motif, 70 PTR, 61, 66, 67, 70 Pulse width, 392

530 Pulsed axial activation, 369 Pulsed double ionization sources, 12 Pulsed dual ion source, 13, 14 Pulsed dual polarity ionization source, 12 Pulsed high pressure mass spectrometry, 389, 396, 398, 402, 406, 409 Pulsed laser, 187, 189 Pulsed Q collision-induced dissociation, PQD, 98, 99 Pulsed triple ionization source, 12 Pulsed-valve method, 44 Purge-and-trap/gas chromatography/mass spectrometry, 491, 492, 494, 496–498, 500–502, 505 Pyridine–pyridine, 397 Pyridine–water, 397

Q qcut-off, 371 QIT, see Quadrupole ion trap QIT Esquire 3000+, Bruker Daltonics, 257, 259, 261, 263, 264, 266, 270, 272, 275, 277, 278, 281, 282 QQQ, see Triple-stage quadrupole qr, 298, 368 QSTAR XL, Applied Biosystems/MDS Sciex, 13 Q-TOF hybrid instrument, 208, 223, 224 QTRAP, 64, 66 Q-Trap 2000, Applied Biosystems/MDS Sciex, 9 qu, 261 Quadratic phase relation, 459 Quadrupole array, 11, 12 Quadrupole bender, 171, 173 Quadrupole ield, quasi-pure, 345 Quadrupole ion trap, 36, 42, 45, 46, 54, 61–64, 67, 70, 87, 88, 90, 96, 97, 101, 107, 190, 207, 208, 211, 240, 244, 253, 255, 258, 260, 262, 328, 339, 367, 369–371 electrodes, cross-section of, 265 instrument, 51, 104, 140 home-built, 186–188 hyperboloidal geometry of, 328 mass spectrometer, 60, 84, 155, 173 modiication for spectroscopy, 257 modiied hyperboloidal angle, 264, 268, 281 non-ideal, 261 Quadrupole mass ilter, 215, 293, 295, 297, 334, 339, 467 Quadrupole mass spectrometer, 398 Quadrupole time-of-light, QTOF, 97 Quadrupole-FT-ICR, 138 Quantiication, 417, 432 Quantum entanglement, 356 Quantum information, 328

Subject Index Quantum jump, 342, 343 number, 360 Quartz crystal, beating, 329, 352 Quasi-stable, 372 QuEcHERS, 476 Quenching measurement, 191, 193 Quenching rate, 191, 194–196 model, 193–195, 197 temperature dependence of, 192–194 Quercetin, 155, 156, 162, 163 Quercetin-3-O-rhamnoside, 156 Quercetin-3-O-rutinoside, 155, 156, 162 Quercitrin, 50 QUISTOR, 328 qz, 173, 174, 191, 259, 261, 268–272, 278, 280–282, 350, 368 qz-axis, 370 qz-value, 446, 465

R Radar, development of, 329 Radial ion ejection, 11 Radial trap frequency, 300 Radiation pressure force, 301 Radiative lifetimes, 169 Radio frequency (RF) cavity, 332 ion trap, 169, 171, 238, 346 Radiofrequency domain, 332 Ramsey fringe, 332 Raster-step size, 422 Rat brain, 423, 424 tissue section, 425 Rate constant, 393, 394 ion/molecule reaction, 398 Rate equation analysis, 192 Rats, control, 432 Rayleigh length, 188 Reactant ion peak, RIP, 395 Reaction kinetics, 36 Reaction rate constant, measurement of, 387, 403, 404, 411 Reaction time, 392, 401 window, 16 Reaction vessel, 12 Rectangular wave voltage, periodic, 374 Rectilinear ion trap, RIT, 42, 44 Reduced mobility, 397, 406 coeficient, 391 K0, 207 Reference measurement, 321 Relative mass resolution, 293, 295 Remotely-controlled instruments, 494 Repeatability, 505 Reporter group ion, 98 Reproducibility, 491, 498 Residence time, 389

Subject Index Residual magnetic ield, 317 Resolution, mass, 441, 467 Resonance ejection, 6, 132 scan, 440, 467 Resonance excitation, 157, 264, 294 /ejection, 382 Resonance frequency, 294, 313 ω+, 304, 305, 307, 317, 322 ω−, 304, 317, 322 Restriction of Hazardous Substances, RoHS, 478, 481, 482 Reversed-phase chromatography, 96, 101 Reversed-phase liquid chromatography, 156, 192 RF barrier, 9 RF circuit, 259, 260 RF drive voltage amplitude, 334, 335 RF electric ield, 334 RF frequency, 298, 307, 314, 322, 341–343, 345–347 RF gain curve, 259 RF ion trap, 188, 294 RF linear trap, 332 RF modulation, 462 RF photon correlation, 342 RF potential, 175, 258, 259, 301, 316, 338, 345, 346, 350 RF power supply, 355 RF ramping, 191 RF unbalance, 9 Rhem–Weller observation, 197 Rhenate, ReO3− and attachment mechanism, 71, 72 Rhodamine 101, 272, 274, 275, 277 Rhodamine 590, 255, 256, 281 Rhodamine 640, 188, 189 Rhodamine 6G, 255 Ring electrode, 7, 191, 258–265, 270, 271, 275, 339, 345, 348, 350, 371, 374 stacked, 208 Ring trap, 347 Ring-down time, 241 Robert A. Welch Foundation, 55 Round rods, 338

S Saccharomyces cerevisiae, 96, 142 Safe Water Drinking Act, SWDA, 468 Sample gas inlet, 390 Sample preparation, 421 Saturn 2000 3D, 449, 467 Saturn 4D, 440 Saturn, Varian, 370 Saturn-I, 440, 467 Saturn-II, 467 Saturn-III, 467

531 Saturn-IV, 467 Scan function, 371, 372, 374 chemical ionization, 449 triple resonance, 442, 467 Scan speed, 443 higher, 439, 442 Scan table, 374, 382 Scan, data-dependent, 464 stepped normalized, 464 Scattering rate, 320 Sciatic nerve, 432 SCSI-MS instrument, sketch of, 294 Second, time unit, 329 Secondary ion mass spectrometry, SIMS, 418 Second-Doppler effect, 332, 334, 339, 354, 359 Secular frequency(ies), 16, 317, 368, 457, 462, 463 Secular frequency, see Fundamental secular frequency summary of calculated values, 267 SELECT, 91, 92, 100 Selected reaction monitoring, SRM, 427 Selective ion monitoring, SIM, 469 Selective ion storage, SIS, 467 Self-CI, see Ionization, self-chemical Sequence coverage, complete, 21 Sequence information, 21 complementary, 21 Serine octomer, conformational structure of, 228 Serum albumin, 134 Servo-loop, 343, 353 scheme, 330 Sewage treatment plant, 503, 505 Shewanella oneidensis, 144 Shuttle trap, 339 Sickle-cell anemia, 222 Side-chain losses, 24 Sigma Aldrich Corporation, 255 Signal-to-noise ratio, S/N, 383, 447 Silver nitrate, 20 SIMION model parameters, 265 SIMION v. 8, Scientiic Instrument Services Inc, 261, 263–268, 273, 278, 345 Simulation trajectories and histograms, 195 Dye–Arg+, 195, 196 Dye–Trp, 195, 196 Trp–Arg+, 195, 196 Single ion oscillation frequency, 302 Single ion preparation, 341 Single ion, sympathetically-cooled, 291, 299, 300 Single trapped ion, 170, 333, 342, 345–347, 355, 357, 360 laser spectroscopy of, 170, 342, 345, 346 Single-ion mass spectrometry, 291, 292, 311, 318

532 Singly-charged anions, 6 Singly-charged ions, large, 17 Skimmer lens, 105 Slides, glass, 431 Slides, indium–tin oxide coated glass, 422 Slides, non-conductive plain glass, 422 Small molecule analysis, 419 Small molecule identiication, 430 S-methyl 5,5′-thiodipentanoylhydroxysuccinimide, 106, 108 structure, 107 SN2 displacement, 400 Sodium, 425 Sodium acetate, 425 Sodium adduct ion, 431, 433 Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE, 101, 154 Sodium ion adduct, 423 Sodium phosphocholine, 427 Solid-phase isotope-labeling strategy, 96 Solvents, chlorinated, 492 Sonic spray ionization, SSI, 11, 62 SORI-CID, 122, 130, 132, 133, 135, 246 principles of, 132, 133 Source region, 105, 387, 389 corona discharge, 389 radioactive nickel foil, 389 ultraviolet discharge lamps, 389 Source, switchable, 447 Space charge, 467 control, 417, 422 interaction, 189 limit, 240 limited density, 174 Space-charging, 423, 424 Spatial resolution, 422 Spatially-resolved measurement, 293 Spectrograph, Shamrock 303i, Andor Technologies, 257 Spectroscopy, action (or consequence), 240, 241 Sphingomyelin, SPM, 425, 432, 433 Spinal cord, 432 Splenic tissue, cryosectioned, 435 Sprague-Dawley rat, 425 Spring constant, 302 Sr+, 355 SSI, 12 Stability diagram, 26, 268, 270, 348, 351, 353, 369–371, 373, 375, 382, 383, 385 boundaries of, 350, 352, 378, 382 computed and experimentally-determined, 380, 381 cross-section of, 351 theoretical, 374 Stability region, 298, 378 irst, 378

Subject Index Stability, instrumental, 505 Stability, reliability, 505 Stable isotope labeling by amino acids in cell culture, SILAC, 94 Stable isotope labeling strategies, summary, 95 Stable trajectory region, 380 Stacked-ring ion guide, SRIG, 210 Stark shift, 359 Stearic acid, 426 Stilbene, 36 Stored waveform inverse Fourier transform, SWIFT, 131, 174–176, 191, 458–460 Streptavidin afinity chromatography, 86 Strong cation exchange (SCX) chromatography, 86 Strontium ion, 346 Structural characterization, 425 Styrene, 495, 497, 500 Sub-Doppler laser cooling, 341 Substatia nigra, 425 Sulfonium ion derivatization, 92 Sulfur dioxide, 6 Sulfur hexaluoride, 407 Supersonic jet, 245 Supplemental ields, dipole, 442, 462, 464 Supplemental ields, quadrupole, 442 Supplemental waveform, 66 Supplementary radiofrequency voltage, 347, 367 Supplementary RF signal, 16 Surface charge, 339 Surface waters, pollution of, 503 Surface-induced dissociation, SID, 130 Sustained off-resonance irradiation CID, see SORI-CID Swarm experiments, 407 Sweep frequencies, 369 Switching circuits, 374 Sympathetic cooling, 292, 300, 356, 360 Sympathetically-cooled single ion mass spectrometry, SCSI-MS, 292, 293, 295, 296, 299, 300, 303, 305, 309, 311, 312, 321, 323, 324 Synapt high deinition mass spectrometer, HDMS, 210 Synapt instrument, 212, 215, 218, 221, 223–230 Synapt, modes of operation, 213 schematic diagram, 211 Syrian Hamster protein, 214 Syrian Hamster PrP protein, 220

T Taggants, 399 Tailored waveform, 16 Tandem mass spectrometric analysis, 96, 97

Subject Index Tandem mass spectrometric strategy, 105 Tandem mass spectrometry, 59, 87, 89, 91, 92, 104, 105, 122, 130, 367, 372, 417, 419, 424–428, 434–436 analysis, 97, 153–164, 214, 224, 226, 230 multiple stages of, 9, 14, 61, 62, 72, 83, 84, 105–107, 133, 212, 226, 367, 425, 426, 430–432, 436 functionality, 25 interrogation scheme, 64 scan type, 61 scan, 100 Tandem-in-space, 454 Tandem-in-time, 5, 454 Tantalum, 345, 346 Tekmar velocity XPT purge and trap, 496 Terahertz (THz) frequency domain, 329 Terephthalic acid, 48 Tetrachloroethylene, 492, 495, 501, 504 Tetrameric transthyretin (TTR) complex, 222 Tetramethylrhodamine, 191 Thermal equilibrium, 388 Thermalization, 389 Thermo Electron Corporation, 420 Thermo Finnigan LCQ mass spectrometer, 42, 43 Thermo Scientiic, 440 Thermo Scientiic ITQ, 447 Thermodyamic data, 387, 391, 394, 397, 398 Three-dimensional (3D) quadrupole ion trap, 5 Tickle activation, 347, 348, 372 Tickle frequency, 348, 349 Tickle voltage, 347, 348, 372 Time metrology, 328, 341, 359 Time-domain signal, 126, 207 Time-of-light mass spectrometer, 293, 295, 419 Time-of-light, TOF, 9, 211 Time-of-light/time-of-light, TOF/TOF, 97 Time-resolved measurement, 293 TiO2, 86 Tissue analysis, 417, 422 Tissue sample, 421, 429 Tissue section(s), 418, 430, 431, 433, 435 Tissue specimens, 424 Tissue studies, 423 Tissue surface, 419 Tissue, intact, 426 ovarian, 419 TMPP-Ac derivative, 90 TMPP-Ac-OSu, 88 TMPP-AcSC6F5 bromide, 88, 89 TOF, 12 TOF mass analyzer, 14, 171, 172, 208 TOF mass spectrum, 174 TOF, orthogonal acceleration, 225 TOF, see Time-of-light mass spectrometer Toluene, 492, 495, 498, 501–504

533 Torus, 345 Total ion chromatogram, TIC, 472, 473, 476–478, 483, 484 Total ion count, TIC, 423 Total organic carbon, TOC, 483 Toxicological drug study, 435 Transcriptional editing process, 59 Transferrin, 141 Transition metal complex cations, 20 Transition metal ion insertion, 20 Transition, luorescence of, 330 Transmission mode ETD, 23 Trap ring aperture, 188 diameter, 188 Trapped ion cloud, 135 Trapped ion electron diffraction, TIED, 169–171, 173 Trapped ion luorescence, 186 spectroscopy, 187, 241 Trapped ion instrument, 60 Trapped ion laser excitation, 187 Trapped ion mass spectrometer, 72, 240–245, 247, 254, 282, 283 Trapped ions, 333 activated by IR laser, 54 dynamics, 169–199, 348 structure, 169–199 Trapping by proxy, 5 Trapping ield imperfection, 316 Trapping ield, hexapolar component of, 263 Trapping frequency calibration, 456 Trapping oscillation, 124 Trapping parameters, 349, 369 Trapping, selective, 456 Traps on micro-chips, 328 Traveling wave IMS, TWIMS, 206, 209, 212, 213, 215–225, 228 Traveling wave ion guide, TWIG, 209–212 Traveling wave, T-Wave, 209–212 Tributylamine, 220 Trichloroethylene, 492, 495, 498, 502–505 Triethylene, 220 Trihalomethanes, 492 Triple-stage quadrupole, 367–369, 427 /LIT mass spectrometer, 11 TriWave, 210, 211, 215 trp RNA binding protein, TRAP, 219 Trp-11 neurotensin, 20 Trp-cage protein charge states, 192 Tryptic lectin glycopeptide, 23 Tryptic peptide, 208 Tryptic protein digest, 219 Tryptophan, Trp, 186, 191, 219 Tumor detection, 419 Turning quadrupole, 6–8 T-wave, 211, 215, 216; see also Traveling wave IMS, TWIMS

534

Subject Index

Two-dimensional differential gel electrophoresis, 2D DIGE, 93 Two-ion crystal, 305 system, 300, 305, 319, 321, 323

vMALDI, see Matrix-assisted laser desorption, intermediate vacuum Volatile organic compounds, VOCs, 492–494, 499, 505

U

W

U.S. Environmental Protection Agency, USEPA, Method, 492 U.S. Naval Observatory, 329 U.S. Navy Observatory, USNO, 354 Ubiquitin, 54, 69, 126, 133 Ultra low expansion, ULE, spacer, 356, 357 Ultra-violet photon dissociation, UVPD, 41 Undersampling, 422 Unipolar mode excitation, 348 Universal constant, 330 Unstable, 368 Upper m/z limit, 14 Urinary metabolites, 208 USEPA Method 505, 484, 485 USEPA Method 521, 439, 470, 472, 481, 483, 484 USEPA Method 524.2, 470 USEPA Method 525.2, 469 USEPA Method 527, 470 USEPA Method 528, 470 USEPA Method 529, 470 USEPA Method 603, 498 USEPA Method 8260, 470 USEPA Method 8260B, 495–497, 505 USEPA Method 8270, 439, 468–470, 473, 475 USEPA Method SW-846, 470 USEPA Methods, 439, 467 USEPA, see U.S. Environmental Protection Agency UV photodissociation, 93

Wannier expression, 391 Wastewater(s), 497, 499, 502, 503 Water, 396, 405 drinking, 483, 492 industrial, 501 reagent, 486 surface, 486 Waters Corporation, 210, 213 Waveform, broadband, 456 Waveform, notch, 456, 457, 460–462 Wine, 153 Wine astringency, 153, 154, 164 Wine tasting, 155 Wine, organoleptic property, 153 Wire mesh, micrometric, 347 Working points, 371

X Xenon cations, 21 X-ray, 173, 221 scattering, 219 Xylene(s), 492, 500

Y Yb+, 355 Yeast enolase, tryptic digest, 91, 92 Ytterbium, 332, 334

Z V Van der Waals complex, 243 Van’t Hoff plot, 396, 399, 400 Varian 4000 ion trap, 442 Varian 4000MS, 449–451, 454 Varian 500MS, 444 Varian quadrupole ion trap mass spectrometer, 494 Varian Saturn, 440 Varian Star 3400X Saturn 2000 GC/MS, 496 Varian turbo DDS, 464 Velocity of light, 330 Vial shield, 450 Vibrational relaxation, 176 Vibrational temperature, 171 Vinblastine, 225 Vinyl chloride, 502 Virus capsids, 220 Virus tails, 220 Viscous damping force, 303, 345

z0, 173, 296 Zeeman effect, 354, 357, 359 Zidovudine, 228 Zoom scan mode, 443 ZrO2, 86 Z-spray source., 210, 211 Zwitterion, 247, 249 α-Helical PrP, 220 α-Helical PrP, β-sheet-rich structures, 220 α-Methylstyrene, 500 β-Casein phosphopeptide, 137 β-Galactosidase, 141 β-Lactoglobulin, 54 βr, 370, 372, 373, 380, 382 βu, 261, 262 βx, 350, 352, 353 βz, 268, 270–272, 276, 350–353, 370, 382, 383, 385 μ-Metal shield, 172