Glycan Microarrays: Methods and Protocols (Methods in Molecular Biology, 2460) 1071621475, 9781071621479

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Methods in Molecular Biology 2460

Michelle Kilcoyne Jared Q. Gerlach Editors

Glycan Microarrays Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Glycan Microarrays Methods and Protocols

Edited by

Michelle Kilcoyne Carbohydrate Signalling Group, Discipline of Microbiology School of Natural Sciences, National University of Ireland Galway, Galway, Ireland

Jared Q. Gerlach Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland

Editors Michelle Kilcoyne Carbohydrate Signalling Group Discipline of Microbiology School of Natural Sciences National University of Ireland Galway Galway, Ireland

Jared Q. Gerlach Advanced Glycoscience Research Cluster School of Natural Sciences National University of Ireland Galway Galway, Ireland

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-2147-9 ISBN 978-1-0716-2148-6 (eBook) https://doi.org/10.1007/978-1-0716-2148-6 © Springer Science+Business Media, LLC, part of Springer Nature 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Glycan microarrays have become a powerful tool to elucidate carbohydrate-mediated binding mechanisms and binding specificity for a wide range of molecules including those that are recombinantly produced or derived from biological fluids and tissues, or in situ as part of microbes or mammalian cells. With increasing awareness of the importance of carbohydrates in a plethora of biological functions and processes, their applications are highly valuable across many disciplines and their use is becoming more and more widespread as their utility becomes better developed by many labs across the world. This book provides a collection of detailed methods for the construction and application of glycan microarrays that will enable researchers to answer questions related to glycobiology and chemical biology. Glycan microarray approaches can be tremendously useful in a myriad of areas including immunology, microbiology, cancer biology, medicine, pharmacology, agriculture and food science, plant biology, vaccine research, and many more. This book is divided into four parts which begin at the basics of glycan microarray construction and progress through the advanced and highly novel applications of glycan and glycomics microarrays. Part I describes the construction of several varieties of glycan microarray platforms and appropriate signal detection methods which can be used with them. Part II follows with microarray data extraction and processing, and then advances to using the generated data to calculate measurements. Part III includes methods for the construction and use of platforms tailored for specific applications, including synthetic plant glycans, natural mucins, bacteria, and tissue microarrays, as well as their bespoke applications. The final section of this book, Part IV, is focused on innovative applications of various glycomics microarrays including autoantibody detection, screening microbial glycans for vaccine development, cancer glycobiomarker discovery, and bacterial and mammalian cell profiling for carbohydrate binding. This valuable resource was made possible by the generosity of leaders and pioneers in the glycomics microarray field who have agreed to share their detailed methods and expertise. We hope this method collection will open up glycan and glycomics microarray methods to an even-greater community of researchers, from fields beyond glycobiology, to further enhance biological insights and to provide meaningful data which leads to breakthroughs in our ability to harness the information contained within the functional glycome. Galway, Ireland

Michelle Kilcoyne Jared Q. Gerlach

v

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

GLYCAN MICROARRAY PLATFORMS

1 Neoglycoprotein and Glycoprotein Printing on a Hydrogel Functionalized Microarray Surface and Incubation with Labeled Lectins. . . . . . . . . . . . . . . . . . . . . Jared Q. Gerlach, Marie Le Berre, and Michelle Kilcoyne 2 Evaluation of Glycan-Binding Specificity by Glycoconjugate Microarray with an Evanescent-Field Fluorescence Detection System . . . . . . . . . . . . . . . . . . . . Lalhaba Oinam and Hiroaki Tateno 3 Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content Analyses of Glycan-Binding Proteins Including Natural Anti-Glycan Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sharad Purohit and Jin-Xiong She 4 Nanocube-Based Fluidic Glycan Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hung-Jen Wu, Akshi Singla, and Joshua D. Weatherston

PART II

3

25

33 45

GLYCAN MICROARRAY DATA EXTRACTION, PROCESSING AND USE FOR MEASUREMENTS

5 General Strategies for Glycan Microarray Data Processing and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Sebastian Temme and Jeffrey C. Gildersleeve 6 Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism . . . . . . . . . . . . . . . . . . . . Marie Le Berre, Jared Q. Gerlach, Iwona Dziembała, and Michelle Kilcoyne

PART III

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67

89

COMPLEX NATURAL AND SYNTHETIC GLYCOCONJUGATE MICROARRAYS

7 Synthetic Plant Glycan Microarrays as Tools for Plant Biology . . . . . . . . . . . . . . . . Colin Ruprecht and Fabian Pfrengle 8 Mucin Purification and Printing Natural Mucin Microarrays . . . . . . . . . . . . . . . . . Marie Le Berre, Jared Q. Gerlach, Mary E. Gallagher, Lokesh Joshi, Stephen D. Carrington, and Michelle Kilcoyne 9 Bacterial Microarrays for Examining Bacterial Glycosignatures and Recognition by Host Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marı´a Asuncio n Campanero-Rhodes and Dolores Solı´s 10 Tissue Glycome Mapping: Lectin Microarray-Based Differential Glycomic Analysis of Formalin-Fixed Paraffin-Embedded Tissue Sections . . . . . . . . . . . . . . . Chiaki Nagai-Okatani, Xia Zou, Atsushi Matsuda, Yoko Itakura, Masashi Toyoda, Yan Zhang, and Atsushi Kuno

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Contents

PART IV APPLICATIONS FOR GLYCOMICS MICROARRAYS 11

12

13

14

15

Detection of Autoantibodies Using Combinatorial Glycolipid Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Susan K. Halstead, Dawn Gourlay, and Hugh J. Willison Glycan Microarrays Containing Synthetic Streptococcus pneumoniae CPS Fragments and Their Application to Vaccine Development. . . . . . . . . . . . . . . . . . . Paulina Kaplonek and Peter H. Seeberger Lectin-Based Protein Microarray for the Glycan Analysis of Colorectal Cancer Biomarkers: The Insulin-Like Growth Factor System . . . . . . . . . . . . . . . . . Dragana Robajac, Martina Krizˇa´kova´, Milosˇ Sˇunderic´, Goran Miljusˇ, Peter Gemeiner, Olgica Nedic´, and Jaroslav Katrlı´k Bacterial Staining and Profiling for Glycan Interactions on Glycan Microarrays for t-Test Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen Cunningham, Jared Q. Gerlach, Andrea Flannery, and Michelle Kilcoyne Preparation and Fluorescent Labeling of Cell-Derived Micelles and Profiling on Glycan Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marie Le Berre, Jared Q. Gerlach, and Michelle Kilcoyne

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors MARI´A ASUNCIO´N CAMPANERO-RHODES • Instituto de Quı´mica Fı´sica Rocasolano, CSIC, Madrid, Spain; CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain STEPHEN D. CARRINGTON • Veterinary Sciences Centre, UCD School of Veterinary Medicine, University College Dublin, Dublin, Ireland STEPHEN CUNNINGHAM • Glycoscience Group, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland IWONA DZIEMBAŁA • Carbohydrate Signalling Group, Discipline of Microbiology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland ANDREA FLANNERY • Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; Carbohydrate Signalling Group, Discipline of Microbiology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland MARY E. GALLAGHER • Veterinary Sciences Centre, UCD School of Veterinary Medicine, University College Dublin, Dublin, Ireland PETER GEMEINER • Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia JARED Q. GERLACH • Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland JEFFREY C. GILDERSLEEVE • Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA DAWN GOURLAY • Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK SUSAN K. HALSTEAD • Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK YOKO ITAKURA • Department of Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan LOKESH JOSHI • Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; Glycoscience Group, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland PAULINA KAPLONEK • Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Potsdam, Germany; Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Berlin, Germany JAROSLAV KATRLI´K • Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia MICHELLE KILCOYNE • Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; Carbohydrate Signalling Group, Discipline of Microbiology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland MARTINA KRIZˇA´KOVA´ • Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia

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Contributors

ATSUSHI KUNO • Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan MARIE LE BERRE • Advanced Glycoscience Research Cluster, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; Carbohydrate Signalling Group, Discipline of Microbiology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland ATSUSHI MATSUDA • Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan; Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan GORAN MILJUSˇ • Institute for the Application of Nuclear Energy (INEP), University of Belgrade, Belgrade, Serbia CHIAKI NAGAI-OKATANI • Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan OLGICA NEDIC´ • Institute for the Application of Nuclear Energy (INEP), University of Belgrade, Belgrade, Serbia LALHABA OINAM • Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan FABIAN PFRENGLE • Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany; Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Vienna, Austria SHARAD PUROHIT • Center for Biotechnology and Genomic Medicine, Augusta University, Augusta, GA, USA; Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta University, Augusta, GA, USA; Department of Undergraduate Health Professionals, College of Allied Health Sciences Augusta University, Augusta, GA, USA DRAGANA ROBAJAC • Institute for the Application of Nuclear Energy (INEP), University of Belgrade, Belgrade, Serbia COLIN RUPRECHT • Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany; Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Vienna, Austria PETER H. SEEBERGER • Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Potsdam, Germany; Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Berlin, Germany JIN-XIONG SHE • Center for Biotechnology and Genomic Medicine, Augusta University, Augusta, GA, USA; Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta University, Augusta, GA, USA AKSHI SINGLA • Department of Chemical Engineering, Texas A&M University, College Station, TX, USA DOLORES SOLI´S • Instituto de Quı´mica Fı´sica Rocasolano, CSIC, Madrid, Spain; CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain MILOSˇ SˇUNDERIC´ • Institute for the Application of Nuclear Energy (INEP), University of Belgrade, Belgrade, Serbia HIROAKI TATENO • Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan J. SEBASTIAN TEMME • Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

Contributors

xi

MASASHI TOYODA • Department of Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan JOSHUA D. WEATHERSTON • Department of Chemical Engineering, Texas A&M University, College Station, TX, USA HUGH J. WILLISON • Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK HUNG-JEN WU • Department of Chemical Engineering, Texas A&M University, College Station, TX, USA YAN ZHANG • Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China XIA ZOU • Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan; Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China

Part I Glycan Microarray Platforms

Chapter 1 Neoglycoprotein and Glycoprotein Printing on a Hydrogel Functionalized Microarray Surface and Incubation with Labeled Lectins Jared Q. Gerlach, Marie Le Berre, and Michelle Kilcoyne Abstract Glycan microarrays are widely used to elucidate carbohydrate binding specificity and affinity of various analytes including proteins, microorganisms, cells, and tissues. Glycan microarrays comprise a wide variety of platforms, differing in surface chemistry, presentation of carbohydrates, carbohydrate valency, and detection strategies, all of which impact on analyte performance. This chapter describes detailed methods for printing neoglycoprotein and glycoprotein microarrays on hydrogel-coated slides and incubation of these glycan microarrays with fluorescently labeled lectins. Key words Glycan microarray, Glycomics microarray, Glycoprotein, Neoglyconjugate microarray, Neoglycoprotein, Microarray printing, Microarray incubation, Lectin profiling, Microarray incubation

1

Introduction Glycan microarrays are widely used to elucidate carbohydrate binding specificity and affinity of various analytes including proteins, microorganisms, cells, and tissues [1–3]. The microarrays themselves comprise a wide variety of platforms, differing in multiple aspects including surface chemistry, presentation of carbohydrates, carbohydrate valency, and detection strategies, and all of these facets impact on the behavior of the analyte [4–7]. Neoglycoproteins are synthetic glycoproteins consisting of a protein carrier or backbone, usually the non-glycosylated human serum albumin (HSA) or bovine serum albumin (BSA), multiply substituted with carbohydrates via a linker, which can also impact on the presentation of the carbohydrate and subsequent analyte behavior [5–7]. The substitution of the carbohydrate moiety on the protein backbone can vary, but key advantages of neoglycoproteins include the known single carbohydrate structural moiety and multivalent presentation of the carbohydrate. Further, the protein

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_1, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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portion of neoglycoproteins and glycoproteins provides a convenient scaffold to immobilize the carbohydrates onto the microarray surface while providing a variety of options for surface conjugation chemistries. Numerous surfaces have been used for glycan microarray platforms, but this chapter describes the use of a 3-dimensional (3D) hydrogel-coated microarray functionalized with N-hydroxysuccinimide activated carboxylic acid functional groups, allowing covalent conjugation via reaction with amine groups of neoglycoproteins and glycoproteins. Several groups, including ours and the Consortium for Functional Glycomics, use a functionalized hydrogel microarray surface due to the inherently low background, lack of necessity for a blocking step for analyte incubations, and increased number of functional groups per unit area in comparison to 2-dimensional (2D) surfaces [4–6]. The conjugation chemistry occurs in the physiological pH range making this platform compatible for conjugating biological molecules without significantly altering or destroying their structure and increasing the likelihood of maintaining molecular functionality after immobilization onto the microarray surface. In this chapter, the construction of a neoglycoprotein and glycoprotein microarray on a hydrogel-coated slide is described, along with incubation of this microarray with a panel of fluorescently labeled lectins.

2

Materials Prepare all solutions in ultrapure water (15.5 MΩ at 25
C) and using analytical grade reagents unless indicated otherwise. Prepare and store all reagents at room temperature unless indicated otherwise. All pipette tips and tubes used should be autoclaved before use or purchased sterilized. Follow all local waste disposal regulations when disposing of waste materials.

2.1 Printing Neoglycoprotein and Glycoprotein Microarray Slides

1. Deionized water (18.2 MΩ at 25
C). 2. 500 mL 0.2 μm filter. We use the Filtropur BT 50 bottle top filter which has a polyethersulfone (PES) filter membrane (catalog number 2023-09-30, Sarstedt, Inc., Newton, NC, USA). 3. Microarray printer. We use a SciFlexArrayer S3 (Scienion AG, Berlin, Germany; catalog no. 1070936) equipped with a 20 slide printing stage and a piezoelectric uncoated 90 μm diameter glass nozzle. 4. Functionalized microarray slides suitable for the desired conjugation reaction. We use Nexterion® slide H microarray slides (Schott AG, Mainz, Germany). The functionalized microarray

Glycan Microarray Printing on Hydrogel Functionalised Surface

5

slides are supplied in packs of 25 in vacuum packed containers and are stored at 20
C until use. 5. Phosphate-buffered saline (PBS), pH 7.4 (see Note 1). Usually 1 L of an autoclaved 10X PBS stock is made and diluted to use concentration (1) with ultrapure water just before use (e.g. 100 mL of 10 PBS added to 900 mL of ultrapure water (15.5 MΩ at 25
C)). 6. A panel of pure neoglycoprotein and glycoproteins of known glycosylation (probes) of relevance for the study (see Note 2). Dissolve in PBS, pH 7.4, to a concentration of 1 mg/mL (based on supplied weight), aliquot 20–50 μL volumes into 500 μL microtubes and store at 20
C until use (see Notes 3 and 4). 7. Microarray printer probe plate. We use the 384-well plate sci SOURCEPLATE 384 PS (catalog number CPG-5501-1, Scienion AG, Berlin, Germany). 8. Saturated sodium chloride (NaCl) solution. In 1 L of ultrapure water, add NaCl with continuous stirring using a stir bar and magnetic plate (approx. 2 g per addition) until the NaCl stops dissolving. 9. Humidity chamber. We use the StainTray slide staining system with the black lid (catalog number Z670146 from SigmaAldrich Co. (now Merck)). It facilitates use as a humidity chamber when a layer (approx. 3 mm depth) of saturated NaCl is poured at the bottom of the chamber and has raised polymer runners to keep slides in place. 10. Five clear glass Coplin jars. 11. Blocking solution: 100 mM ethanolamine in 50 mM sodium borate, pH 8.0. To approximately 700 mL ultrapure water, add 3.0 g boric acid (99.5% purity, catalog number B0252, Sigma-Aldrich Co.) with stirring. Add several drops of 50% NaOH to the solution with constant stirring until the sodium borate dissolves. Add 6 mL of ethanolamine (99.0% purity, catalog number 398136-25ML, Sigma-Aldrich Co.) to the sodium borate solution. Adjust the pH to 8.0 using concentrated HCl (dropwise) with constant stirring. Make up to 1 L with ultrapure water. Make blocking solution fresh just before use. Approximately 500 mL is required to block 20 slides. 12. PBS-T: 100 mL 10 PBS and add 900 mL ultrapure water and 0.5 mL molecular biology grade Tween® 20 (0.05%) (see Note 5). 13. 50 mL sterile polypropylene tubes. 14. Flat topped plastic tweezers.

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15. Slide box to store the printed microarray slides. We usually re-use the slide box in which the functionalized microarray slides were supplied. 16. Desiccant for storage of printed glycan microarray slides. We usually use dry calcium chloride (CaCl2) powder as a desiccant in a polypropylene tube with a pierced cap. 17. Resealable plastic bags. 2.2 Incubation of Labeled Lectins on Neoglycoprotein and Glycoprotein Microarray Slide

1. A low salt version of Tris-buffered saline supplemented with Ca2+ and Mg2+ ions (TBS) is used for glycomics microarray experiments (see Notes 6 and 7). TBS: 20 mM Tris–HCl, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH to 7.2 with concentrated HCl. Usually 1 L of a 10 TBS stock is made, autoclaved, and diluted to 1 with ultrapure water just before use. 2. TBS-T: 100 mL 10 TBS and add 900 mL ultrapure water and 0.5 mL molecular biology grade Tween® 20 (0.05%) (see Note 5). 3. A selection of tetramethylrhodamine isothiocyanate- (TRITC)labeled lectins (Table 1) appropriate for print validation (see Notes 8 and 9). Stock solutions from commercial vendors are typically supplied at 1 or 5 mg/mL (see Note 10). Dilute each fluorescently labeled lectin to 5 μg/mL in TBS-T (see Note 11) 30 min before use and store on ice until use (approximately 100 μL of each fluorescently labeled lectin solution is required for one microarray slide). Fluorescently labeled lectins should always be handled in the dark and dilutions should be prepared and held in the dark. 4. One neoglycoprotein and glycoprotein microarray slide.

Table 1 A selection of lectins commonly used for printing validation in our laboratory, their sources, and carbohydrate binding specificities Lectin

Source

Carbohydrate binding specificity

Con A

Canavalia ensiformis

Man in high-mannose type, hybrid type, and biantennary complex type N-glycans

GS-II

Griffonia simplicifolia (lectin II)

Terminal GlcNAc

AIA

Artocarpus integrifolia

(Neu5Ac)Gal-β-(1 ! 3)-GalNAc-α-O-S/T (T-antigen)

WGA

Triticum vulgaris

GlcNAc-β-(1 ! 4)-GlcNAc-β-(1 ! 4)-GlcNAc, sialic acid

UEA-I

Ulex europaeus

Fuc-α-(1 ! 2)-Gal-R

MAA

Maackia amurensis

Neu5Ac/Gc-α-(2 ! 3)-Gal-β-(1 ! 4)-GlcNAc-β-(1 ! R

SNA-I

Sambucus nigra

Neu5Ac-α-(2 ! 6)-Gal(NAc)-R

Glycan Microarray Printing on Hydrogel Functionalised Surface

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5. Sterile or autoclaved 500 μL microcentrifuge tubes. 6. Gasket slides. We use 8-well gasket slides (catalog number G2534-60015, Agilent Technologies, Inc., Santa Clara, CA, USA). 7. Metal incubation (hybridization) chamber assembly (one chamber per microarray slide to be incubated). We use catalog number G2534A-60000 from Agilent Technologies, Inc. 8. Hybridization oven with rotating arms suitable for accommodating the incubation chamber assembly. We use the microarray hybridization oven from Agilent Technologies, Inc. (catalog number G2545A). 9. Thick walled glass staining dish. We use a 120  120  50 mm staining dish with a fitted glass lid. 10. Flat topped plastic tweezers. 11. Clear glass Coplin jar. 12. 50 mL sterile polypropylene tubes. 13. Microarray slide holders. We use Agilent slide holders for the G2505B microarray scanner (catalog number G2505-60525). 14. Microarray scanner with appropriate laser(s) for fluorescent label(s). We use an Agilent G2505B DNA microarray scanner equipped with 532 nm (green) and 633 nm (red) lasers. 15. 0.1% sodium dodecyl sulfate (SDS) (aqueous).

3

Methods

3.1 Printing Neoglycoprotein and Glycoprotein Microarray Slides

Starting up the microarray printer (Subheading 3.1.1) and steps 1– 5 (Subheading 3.1.2) should be carried out in parallel to increase efficiency.

3.1.1 Starting up Microarray Printer

1. Empty the waste water container and fill fresh water container to just over half way with ultrapure water (Fig. 1a). Turn on the probe plate chiller and set to 10
C (Fig. 1a). 2. Filter approximately 1.1 L of 18.2 ΩM water through the 0.2 μm PES bottle top filter membrane using a vacuum filtration system attached to a 1 L glass system liquid bottle (Fig. 1b). Leave the filtered water under vacuum for 15–20 min to allow it to completely degas (see Note 12). 3. Detach the vacuum attachment from the bottle top filter system and turn off the vacuum. Unscrew the bottle top filter system from the system liquid bottle. Pour off approximately 100 mL of the degassed 18.2 ΩM water and retain a portion (up to 15 mL) in a tube for cleaning the printer nozzle after the

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Fig. 1 Photographs of (a) printer waste and fresh water containers and probe plate chiller, (b) system liquid bottle containing fresh degassed and filtered 18.2 ΩM water with attached Filtropur BT 50 (Sarstedt) bottle top filter, and (c) Scienion SciFlexArrayer S3 microarray printer housing containing probe plate holder, printing stage for microarray slides, and printer nozzle

microarray printing run. Attach the system liquid bottle with the filtered, degassed water (approximately 1 L) to the microarray printer. 4. Turn on the microarray printer followed by the operating computer. Insert the appropriate nozzle into the printer (see Note 13). Complete the startup protocols as per manufacturer’s instructions to attach the nozzle to the pump, clean the printer lines, and adjust voltage and frequency to create a stable printer drop using the system liquid (water).

Glycan Microarray Printing on Hydrogel Functionalised Surface 3.1.2 Printing Neoglycoprotein and Glycoprotein Microarray Slides

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1. Remove a pack of vacuum sealed Nexterion® slide H microarray slides from the freezer and leave to thaw on the bench at room temperature for 30 min before use. 2. Remove aliquots of desired neoglycoproteins and glycoproteins from the freezer and allow to thaw on ice. Once thawed, centrifuge the aliquots for 1 min at 6000 rpm (3,824  g) to deposit any particles or precipitates at the bottom of the microtubes (see Note 14). Remove the microtubes carefully from centrifuge and place in a plastic rack in print order. Leave the plastic rack on ice. 3. Pipette 15 μL of each neoglycoprotein and glycoprotein aliquot into the probe printing plate (Fig. 2a) in desired printing order. Make sure to pipette from the middle of the aliquot and not near any precipitate. 4. Place the lid on the probe printing plate and centrifuge the plate at 1500 rpm (168  g) for 2 min to pull the liquid

Fig. 2 Photographs of (a) microarray printer probe plate, (b) probe plates balanced in a centrifuge, (c) Scienion SciFlexArrayer S3 microarray printer housing containing probe plate and microarray slides inserted onto the printing stage, and (d) printed microarray slides placed in the humidity chamber (slide staining chamber with a layer of saturated NaCl solution at the bottom of the chamber)

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aliquots to the bottom of each well and remove any air bubbles (Fig. 2b) (see Note 15). Carefully remove the probe plate from the centrifuge, making sure not to disturb the liquid in the wells, and insert the probe plate onto the plate chiller in the microarray printer housing (Figs. 1c and 2c). 5. Open the sealed pack of microarray slides and fit 20 microarray slides into the individual places on the printer stage (Fig. 2c) (see Notes 16–19). Make sure to place the slides with functionalized surface facing up and the barcode orientated in the correct direction (following the slide map in the operating software) (Fig. 3a) (see Note 20). 6. Once the slides are loaded, remove the lid from the probe printing plate and close the printer housing door. 7. Set the printer humidifier to 62% (+/2%) and make sure the room temperature is set to 20
C (see Note 21). Following the manufacturer’s instructions for the operating software, load the desired .gal file for printing and select the locations of microarray slides to be printed on the printer stage (Fig. 3b). Start the print run.

a Field 1

Field 2

Field 3

Field 4

Field 5

Field 6

Field 7

Field 8

b

Fig. 3 Maps of (a) probe printing on microarray slides, and (b) microarray printing stage with highlighted (light green) locations to be printed

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8. After the print run is completed (see Note 22), pour a layer of saturated sodium chloride into the slide staining chamber with a lid to create a humidity chamber. Remove the printed microarray slides from the printer stage one at a time and place the slides in the humidity chamber with printed side facing upwards (Fig. 2d) (see Note 17). Place the lid on the chamber and incubate the slides in the humidity chamber overnight at room temperature (20
C) to facilitate probe conjugation. Do not allow the microarray slides to get wet. 9. After the overnight incubation, place the microarray slides in dry clean glass Coplin jars, four slides to a jar (Fig. 4a). Make sure the microarray slides are placed barcode side at the top of the Coplin jar to facilitate handling (as the barcode is not printed with probes and is available to grab the slide) and that the printed sides of the microarray slides do not touch the sides of the Coplin jar or each other. 10. Pour blocking solution into the Coplin jars by tilting the jar at approximately a 45
angle and pouring the blocking solution onto the wall of the jar with the slides perpendicular to that wall (Fig. 4b) (see Note 23). Slowly bring the jar upright while filling with blocking solution to cover the entire microarray slide. Place the lids on the Coplin jars and incubate static on the bench for 1 h at room temperature (see Note 24). 11. From this point, never allow the microarray slides to dry until they are dried evenly by centrifugation. After the blocking incubation, pour off the blocking solution by pouring from the side of the Coplin jar perpendicular to the microarray slides (Fig. 4c). Place a gloved finger over the mouth of the Coplin jar when pouring (but do not touch the slides) to ensure that if

Fig. 4 Photographs of (a) printed microarray slides placed in a clear glass Coplin jar, (b) pouring blocking or washing solution into the loaded Coplin jar by pouring the solution against the side wall of the jar perpendicular to the microarray slides, and (c) pouring off blocking or washing solution from the microarray slide loaded Coplin jar

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any microarray slide is dislodged during pouring, it will not fall out of the jar. Immediately fill the Coplin jar with PBS-T with a volume sufficient to cover the microarray slides by pouring the PBS-T against the side of the jar as described in step 11 above. 12. Replace the lid on the Coplin jar and place it on a plate shaker. Shake the jar gently for 5 min and pour off the PBS-T. This is one wash. 13. Immediately after pouring off the PBS-T wash, fill the Coplin jar with PBS-T as described in step 11 and wash with gentle shaking as per step 12. 14. Repeat the wash steps 11 and 12 three times in total. After the last PBS-T wash, fill the Coplin jar with PBS as described in step 11 and wash with gentle shaking as per step 12. Do not pour off the last PBS wash. 15. Using a plastic tweezers to grip the microarray slides at the barcode, transfer the blocked and washed microarray slides to clean and dry 50 mL polypropylene tubes, one slide per tube with the barcode side at the top of the tube (Fig. 5). Screw the cap onto the tube as soon as each microarray slide is placed in the tube to avoid uneven drying on the slide surface. 16. Centrifuge the 50 mL tubes at 1500 rpm (475  g) for 5 min to dry the microarray slides (see Note 25). After centrifugation, remove the tubes from the centrifuge carefully to avoid disturbing the liquid at the bottom of the tubes and re-wetting the dried slides. 17. Unscrew the caps from the tubes and remove the microarray slides from the tubes using a plastic tweezers to grip the barcode side of the microarray slide. When the microarray slide is free of the tube, grasp the sides using fingers to give better control to hold the slide (Fig. 6a). Handling the slide only by their edges, place the dried printed microarray slides into a slide box (Fig. 6b). 18. Place the printed microarray slide box with desiccant (see Note 26) in a resealable clear plastic bag or container and store at 4
C until use (see Note 27). One slide from each microarray print batch should be validated for intact feature and correct structure presentation before use by incubating with a panel of fluorescently labeled lectins (see Subheading 3.2 below). 3.2 Incubation of Labeled Lectins on Neoglycoprotein and Glycoprotein Microarray Slide

1. All reagent preparation and microarray incubation steps are carried out at room temperature in the dark unless otherwise indicated (see Note 28). A dark room equipped with a red safelight is suitable for all following steps. 2. Remove the microarray slide box still in its resealable bag with desiccant (Fig. 6c) from the 4
C fridge and leave on the bench

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Fig. 5 Photographs of (a) transferring the blocked and washed printed microarray slide from the final wash solution in the Coplin jar to a clean, dry 50 mL polypropylene tube using a plastic tweezers to grip the barcode side of the microarray slide, (b) placing the microarray slide into the polypropylene tube with the barcode side of the slide orientated to the top of the tube, and (c) microarray slides orientated correctly in polypropylene tubes before the tubes are capped for drying by centrifugation

at room temperature for 30 min before use. This allows the microarray slide to equilibrate to room temperature and avoids condensation forming on the slide before use. 3. Turn on the hybridization oven and set to 23
C (or desired incubation temperature) to allow it to reach the required temperature for microarray incubation.

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Fig. 6 Photographs of (a) removing dried microarray slides from polypropylene tubes using a plastic tweezers, (b) placing the microarray slide into a slide box, and (c) a slide box with CaCl2 in a polypropylene tube as the desiccant stored in a resealable plastic bag

4. Disassemble a metal incubation chamber and place an 8-well gasket slide (Fig. 7a) in the appropriate cavity in the lower part of the chamber (Fig. 7b). Pipette 70 μL of 5 μg/mL of each fluorescently labeled lectin (Table 1) diluted in TBS-T in the middle of each well, one lectin per well (Fig. 7c) (see Note 29). In one well, pipette 70 μL of TBS-T (see Note 30). Make sure to note in which well each sample (lectins and TBS) has been placed on the gasket slide. This information is critical to correctly assign signals from each subarray in the resulting microarray data.

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Fig. 7 Photographs of (a) metal hybridization chamber assembly (left) and 8-well gasket slide (right), (b) chamber assembly disassembled and gasket slide inserted into chamber, and (c) 70 μL of each sample pipetted into the middle of each appropriate well on gasket slide

5. Remove the slide box from the sealed plastic bag with desiccant. Take one microarray slide from the slide box and sandwich the microarray slide on top of the 8-well gasket slide (Fig. 8a–d) (see Note 31). Immediately assemble the incubation chamber and tighten the screw of the chamber assembly top to finger tight (Fig. 8e–f). 6. Insert the assembled incubation chamber into the hybridization oven pre-set to 23
C. Close the door and incubate for 1 h with gentle rotation (4 rpm) (Fig. 9a). 7. Ten minutes before the incubation is finished, three quarters fill the staining basin with TBS-T and fill a Coplin jar with TBS-T.

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Fig. 8 Sandwiching the microarray slide with the samples on the gasket slide and assembly of the incubation chamber. (a) Place the top of the microarray slide (slide end furthest from the barcode) in the top of the lower chamber (the side furthest from the barcode end of the gasket slide) such that the printed side of the microarray slide faces the samples pipetted onto the gasket slide (i.e., when the slides are in contact with one another, the printed probes will be available for interaction with the sample). The slide should be hinged by the lower chamber and freely movable up and down using one finger placed at the bottom (barcode end) of the slide as pictured. (b, c) Slowly and smoothly lower the microarray slide on top of the gasket slide. Do not pause or reverse movement. (d) When the microarray slide is at the bottom of its arc, let go of the slide. (e) Immediately assemble the incubation chamber. (f) Tighten the screw (finger tight). Microarray slide is now ready for incubation in the hybridization oven

8. After incubation, remove the assembled incubation chamber from the hybridization chamber and disassemble on the bench. From the lower part of the chamber, remove the gasket and microarray slides which are now adhered together, being careful to always handle slides by their sides only (Fig. 9b).

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Fig. 9 Photographs of (a) sealed incubation chamber placed in incubation oven, and (b–g) the process to remove and release the microarray slide after incubation and wash it. (e) Arrow points to the released gasket slide allowed to fall to the bottom of the filled staining dish. (g) Arrow points to the microarray slide in the filled Coplin jar with the barcode end of the slide orientated towards the top of the jar

9. Submerge the sandwiched slides in the three quarters filled staining basin, making sure to hold the sandwiched slide at their sides (not their ends) with the gasket slide at the bottom (Fig. 9c). Keeping the sandwiched slides submerged, insert the flat head of the lower prong of the plastic tweezers between the two slides at the barcode end and twist the tweezers to release the slides from one another (Fig. 9d). Allow the gasket slide to fall to the bottom of the staining dish while keeping hold of the upper microarray slide (Fig. 9e). From this point, never allow the microarray slide to dry until it is dried evenly by centrifugation.

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10. Remove the microarray slide from the staining dish and immediately insert into a Coplin jar filled with TBS-T, ensuring that the barcode end of the microarray slide is orientated towards the top of the jar to facilitate handling (Fig. 9f). Place the lid on the Coplin jar, place the Coplin jar onto a plate shaker, and shake the jar gently for 5 min. 11. Pour off the TBS-T wash by pouring from the side of the Coplin jar perpendicular to the microarray slides (Fig. 4c). Place a gloved finger over the mouth of the Coplin jar when pouring (but do not touch the slides) to ensure that if any microarray slide is dislodged during pouring, it will not fall out of the jar. This is one wash. 12. Immediately fill the Coplin jar with TBS-T with a volume sufficient to cover the microarray slides by pouring the TBS-T against the side of the jar. Tilt the jar at approximately a 45
angle and pour the blocking solution onto the wall of the jar with the slides perpendicular to that wall (Fig. 4b) (see Note 23). Slowly bring the jar upright while filling with TBS-T to cover the entire microarray slide. Place the lid on the Coplin jar, place the Coplin jar onto a plate shaker, and shake the jar gently for 5 min. 13. Repeat steps 11 and 12 for a third wash in TBS-T. 14. After pouring off the third TBS-T wash, immediately fill the Coplin jar with TBS as described in step 12 (see Note 32). Place the lid on the Coplin jar, place the Coplin jar onto a plate shaker, and shake the jar gently for 5 min. Do not pour off the last TBS wash. 15. Remove the Coplin jar from the plate shaker and remove the lid. Transfer the microarray slide from the wash solution in the Coplin jar to a clean dry 50 mL polypropylene tube by gripping the barcode end of the slide with plastic tweezers and grabbing the side of the slide with fingers (Fig. 10a). Place the slide in the tube orientated with the barcode end to the top of the tube to facilitate handling. Cap the tube immediately (Fig. 10b). 16. Dry the microarray slide by centrifugation at 1500 rpm (475  g) for 5 min at 15
C (see Notes 25 and 33). 17. While the microarray slide is drying, turn on the microarray scanner instrument computer and start the software as per manufacturer’s instructions. Turn on the instrument after software startup. The instrument lasers take approximately 5 min to warm up before use. 18. After slide centrifugation, remove the microarray slide from the tube using plastic tweezers and gripping the side of the slide with fingers, being careful to not disturb the liquid at the bottom of the tube. Open a slide holder cover (Fig. 10c) and

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Fig. 10 Photographs of (a) placing the incubated and washed microarray slide in the polypropylene tube while holding the slide at the sides and orientating the slide with the barcode end upwards, (b) the microarray slide in the capped polypropylene tube before centrifugation, (c) microarray slide holder, (d) placing the microarray slide into the open slide holder, (e) closing the slide holder with the microarray slide inside, and (f) placing the microarray slide in the microarray scanner carousel before scanning

place the microarray slide in the slide holder supported by the ledges inside (Fig. 10d). The microarray slide must be orientated so that the printed side of the slide faces up (i.e., will be covered by the slide holder cover when it is closed) and the barcode end of the slide is furthest away from the hinge (i.e., the barcode will be visible when the slide holder cover is closed). Close the slide holder, taking care not to touch the slide at all (Fig. 10e) (see Note 34). 19. Handling the slide holder by its sides and not touching the microarray slide, transfer the slide holder into the microarray carousel (Fig. 10f). Close the lid of the microarray scanner, select the appropriate settings (e.g., green (532 nm) laser for TRITC label, 90% laser power, 5 μm resolution), and scan the microarray slide. The resulting image will be digitally saved as a .tif image file.

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20. Signal intensity values can be extracted from the .tif file using appropriate microarray feature extraction software (e.g., GenePix Pro v6.1.0.4 (Molecular Devices, Berkshire, UK) and the . gal file for the microarray slide print. 3.3 Washing Gasket Slides for Re-use

1. Gasket slides can be cleaned and re-used up to 20 times. Transfer the released gasket slide from the TBS-T basin to a sterile 50 mL polypropylene tube using a plastic tweezers to hold the barcode end of the slide. Fill the polypropylene tube with approximately 40 mL 0.1% SDS, cap the tube and placed on a rotator (approximately 20 rpm) for 30 min (see Note 35). 2. Wash the gasket slides in ultrapure water four times by emptying the SDS solution, filling the tube with ultrapure water, capping the tube, inverting three or four times and emptying the tube. 3. After the last wash, empty the tube of liquid, cap the tube, and dry the gasket by centrifuging the tube at 1500 rpm (475  g) for 5 min (see Note 25). Remove the tube from the centrifuge carefully to avoid disturbing the liquid at the bottom and to keep the gasket slide dry. 4. Remove the gasket slide from the tube with tweezers, while being careful not to re-wet the slide with the liquid remaining at the bottom of the tube. Place the clean and dried gasket slide in slide storage box and store at room temperature until re-use.

4

Notes 1. Correct print buffer pH is critical for successful conjugation of glycoproteins and neoglycoproteins to the functionalized microarray surface. 2. Pure neoglycoproteins and glycoproteins are available from many international reagent companies including SigmaAldrich Co. (Merck) (Dublin, Ireland), IsoSep AB (Tullinge, Sweden), Dextra Laboratories Ltd. (Reading, UK), and Elicityl (Crolles, France). This list is not exhaustive. 3. Stocks can be made, aliquoted (e.g., 20–50 μL aliquots in 500 μL microtubes) and stored at 20
C. Aliquots can be removed and thawed on ice just before. We have stored neoglycoprotein and glycoprotein stocks at 1 mg/mL in PBS pH 7.4 for up to 2 years at 20
C with no noticeable impact on performance. 4. Usually neoglycoproteins and glycoproteins are supplied from the manufacturer as a lyophilized powder of determined weight with no residual salts. However if residual salts are present and contain primary amine or ammonium ions, these must first be

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removed by buffer exchange with PBS, pH 7.4, or water. Otherwise the protein will not conjugate well to the functionalized microarray surface due to the amines from the buffer solution interfering with the conjugation reaction. 5. Tween® 20 is moderately viscous. Maintain a very slow draw when pipetting and dispensing the detergent or use a positive displacement pipette if available. Invert the PBS-T to mix after addition of the detergent and allow at least 15 min for the detergent to dissolve in the PBS before use. 6. We (and others) use a combination of Ca2+ and Mg2+ for functional lectin applications as many lectins require the presence of these divalent cations to function correctly. Mn2+ can also be included in the lectin buffer but this ion precipitates from TBS within a few hours and can leave debris on slides and interfere with imaging so we typically do not include it in our 10 TBS buffers. If Mn2+ is required for the experiment, it can be added to the 1 TBS buffer just before use. 7. We do not recommend using PBS for experiments involving lectin binding. Some lectins require metal ions for their carbohydrate binding function and phosphate in PBS complexes the Ca2+ into insoluble precipitates, so that the Ca2+ is no longer available for use by the lectin. PBS can decrease or abolish the function of some lectins. 8. A fluorescent label compatible with the microarray scanner in used should be selected. Fluorescein isothiocyanate (FITC) and TRITC are common commercially available fluorescent label options for lectins. 9. Lectins with various fluorescent labels are commercially available from many international reagent companies including EY Laboratories Inc. (San Mateo, CA, USA), Sigma-Aldrich Co., Vector Laboratories (Burlingame, CA, USA), Elicityl (Crolles, France), and Thermo Fisher Scientific. This list is not exhaustive. 10. We recommend making sterile 20 μL aliquots of fluorescently labeled lectins upon receipt and storing frozen at 20
C for long-term storage (months to years) or at 4
C for use in the short term (days to weeks). Aliquots must be protected from light at all times. Do not refreeze aliquots from 20
C once thawed. We have used fluorescently labeled lectin solutions for up to 1 year stored continuously at 4
C and used under sterile conditions. We have used aliquots from 20
C which were stored for up to 3 years after initial aliquoting. 11. 5 μg/mL is a typical concentration used by our lab for fluorescently labeled lectin validation of neoglycoprotein and glycoprotein microarray print but optimal concentrations should be titrated for specific samples.

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12. At intervals tap the side of the glass bottle using a metal spatula. When gas is still present, the tapping will disturb gas bubbles and release them from the liquid. After the water is fully degassed, tapping the glass bottle will not release any gas bubbles. 13. The glass nozzle diameter and coating impacts the resulting feature diameter and must be optimized for individual microarray printer instrument, microarray chemistry, and selected probes. In a typical neoglycoprotein and glycoprotein microarray print in our lab, we use an uncoated 90 μm glass nozzle to produce drops of approximately 450 pL. We print two drops per feature (i.e., approximately 900 pL probe per feature) which results in circular features of approximately 230 μm). 14. Probes which precipitate out of solution are not suitable for printing. Remove from probe set. 15. Make sure to include a balance plate in the centrifuge (Fig. 2b). 16. Load the slides from the back to the front of the printing stage to avoid sleeves or hands touching the slide surfaces. 17. Only ever handle the microarray slides by their sides, both before and after printing. Never touch the microarray slide surfaces to avoid leaving permanent marks and deactivating functional coating. 18. Functionalized microarray slides should only be loaded when printer is ready to print. Do not leave functionalized slides to sit for long periods as the functional groups will be deactivated by hydrolysis from environmental humidity. 19. Slide H microarray slides are supplied vacuum-packed in packs of 25 slides. As the microarray printer will only accommodate 20 microarray slides at a time, the 5 excess microarray slides can be vacuum sealed and stored at 20
C for a subsequent print. If a vacuum sealer is not available, the excess microarray slides may be kept in their slide box, the slide box placed in a resealable plastic bag and sealed with a desiccant (CaCl2 in polypropylene tubes as described in Note 26 below), and stored at 20
C. Once opened, functionalized microarray slides start to lose their functional groups to hydrolysis. Excess microarray slides stored in the latter manner should be used within 48 h. 20. Double check that all slides are securely fitted into their housings (springs) on the printing stage and are lying flat and even on the printing stage surface. A microarray slide that extends up unevenly will smash the nozzle during printing. 21. Microarray printing should be carried out in a room with temperature and humidity control as these properties are critical to achieving a successful print. We recommend maintaining a humid atmosphere of 50–67% and a room temperature of

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20
C (depending on local environmental and optimal print conditions including choice of microarray functionalization, nature of probes, and conjugation chemistry used). We have observed that humidity above approximately 69% and temperatures above 23
C causes feature spreading and decreased probe conjugation on the microarray surface due to loss of microarray surface functional groups to competitive hydrolysis. Additionally, higher temperatures decrease the retained functionality of any printed functional proteins. 22. Print run times vary depending on number of slides to be printed, number of probes and replicate features, and number of subarrays. Our typical print of 20 microarray slides of 52 probes in 6 replicate features each per subarray and 8 replicate subarrays per slide takes approximately 9.5 h. 23. Do not pour liquid directly on the printed side or face of the slide as tidemarks may be introduced. 24. The blocking step reacts any remaining functional groups on the microarray slide surface with ethanolamine to deactivate them and ensure that no other molecules are inadvertently conjugated to the microarray slide surface during incubations. 25. Make sure to balance the tubes in the centrifuge. 26. We usually use CaCl2 in a polypropylene tube as a desiccant. The tube cap is pierced to allow air exchange and facilitate the desiccant absorbing moisture and the mouth of the tube is covered with a thin low shed tissue (e.g., Kim wipe) to keep the CaCl2 powder in the tube. 27. We have used neoglycoprotein and glycoprotein microarrays stored under these conditions for up to 4 years with no apparent impact on their function. 28. Fluorescent labels are bleached if exposed to light. In our experience, exposure to light during reagent preparation, incubation, or just before scanning is the most common reason that no or low fluorescent signal is observed in microarray experiments. 29. Pipette the sample in the middle of well on the gasket slide. Do not allow the sample to touch against the rubber at the edge of well as there may be leakage from the well when sandwiching the microarray slide into place. Do not pipette any bubbles into the sample. If a bubble forms, touch gently with a clean needle or pipette tip to remove it. 30. The resulting signal from the microarray subarray incubated with TBS-T is from probe autofluorescence. Typically these signals are negligible (100 μM), the lectins should be cross-linked with antibody prior to incubation with glycoconjugate microarray to detect their weak glycan-binding activity by cluster effect. 3. If the lectins are expected to contain inactive forms of lectins, try this method. References 1. Boyd WC, Shapleigh E (1954) Specific precipitating activity of plant agglutinins (lectins). Science 119:419. https://doi.org/10.1126/ science.119.3091.419 2. Blixt O, Collins BE, van den Nieuwenhof IM et al (2003) Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelinassociated glycoprotein. J Biol Chem 278: 31007–31019. https://doi.org/10.1074/jbc. M304331200 3. Mega T, Hase S (1991) Determination of lectin-sugar binding constants by microequilibrium dialysis coupled with high performance liquid chromatography. J Biochem 109: 600–603 4. Shinohara Y, Kim F, Shimizu M et al (1994) Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor based on surface plasmon resonance. Eur J Biochem 223:189–194. https://doi. org/10.1111/j.1432-1033.1994.tb18982.x 5. Dam TK, Gerken TA, Cavada BS et al (2007) Binding studies of alpha-GalNAc-specific lectins to the alpha-GalNAc (Tn-antigen) form of porcine submaxillary mucin and its smaller fragments. J Biol Chem 282:28256–28263 6. Tateno H, Nakamura-Tsuruta S, Hirabayashi J (2007) Frontal affinity chromatography: sugarprotein interactions. Nat Protoc 2:2529–2537. https://doi.org/10.1038/nprot.2007.357 7. Blixt O, Head S, Mondala T et al (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci U S A 101:17033 LP–17038 LP. https://doi.org/10.1073/ pnas.0407902101 8. Yan M, Zhu Y, Liu X et al (2019) Nextgeneration glycan microarray enabled by DNA-coded glycan library and nextgeneration sequencing technology. Anal Chem 91:9221–9228. https://doi.org/10. 1021/acs.analchem.9b01988 9. Paulson JC, Blixt O, Collins BE (2006) Sweet spots in functional glycomics. Nat Chem Biol

2:238–248. https://doi.org/10.1038/ nchembio785 10. Tateno H, Mori A, Uchiyama N et al (2008) Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycanbinding proteins. Glycobiology 18:789–798. https://doi.org/10.1093/glycob/cwn068 11. Hu D, Tateno H, Sato T et al (2013) Tailoring GalNAcα1-3Galβ-specific lectins from a multispecific fungal galectin: dramatic change of carbohydrate specificity by a single amino-acid substitution. Biochem J 453:261–270. https://doi.org/10.1042/BJ20121901 12. Kanemaru K, Noguchi E, Tahara-Hanaoka S et al (2019) Clec10a regulates mite-induced dermatitis. Sci Immunol 4:eaax6908. https:// doi.org/10.1126/sciimmunol.aax6908 13. Ogawa T, Sato R, Naganuma T et al (2019) Glycan binding profiling of jacalin-related lectins from the Pteria penguin pearl shell. Int J Mol Sci 20:1–14. https://doi.org/10.3390/ ijms20184629 14. Sakai K, Hiemori K, Tateno H et al (2018) Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation. Med Mycol 57:71–83. https://doi.org/10.1093/mmy/myx163 15. Unno H, Nakamura A, Mori S et al (2018) Identification, characterization, and X-ray crystallographic analysis of a novel type of lectin AJLec from the sea anemone Anthopleura japonica. Sci Rep 8:11516. https://doi.org/ 10.1038/s41598-018-29498-0 16. Sato T, Tateno H, Kaji H et al (2017) Engineering of recombinant Wisteria floribunda agglutinin specifically binding to GalNAcβ1,4GlcNAc (LacdiNAc). Glycobiology 27:743–754. https://doi.org/10.1093/ glycob/cwx038 17. Itakura Y, Nakamura-Tsuruta S, Kominami J et al (2017) Sugar-binding profiles of chitinbinding lectins from the hevein family: a comprehensive study. Int J Mol Sci 18:1160. https://doi.org/10.3390/ijms18061160

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18. Dion J, Advedissian T, Storozhylova N et al (2017) Development of a sensitive microarray platform for the ranking of galectin inhibitors: identification of a selective Galectin-3 inhibitor. Chembiochem 18:2428–2440. https:// doi.org/10.1002/cbic.201700544 19. Shimokawa M, Haraguchi T, Minami Y et al (2016) Two carbohydrate recognizing domains from Cycas revoluta leaf lectin show the distinct sugar-binding specificity—a unique mannooligosaccharide recognition by N-terminal domain. J Biochem 160:27–35. https://doi.org/10.1093/jb/mvw011 20. Unno H, Matsuyama K, Tsuji Y et al (2016) Identification, characterization and X-ray crystallographic analysis of a novel type of mannose-specific lectin CGL1 from the Pacific oyster Crassostrea gigas. Sci Rep 6:29135. https://doi.org/10.1038/srep29135 21. Kenmochi E, Kabir SR, Ogawa T et al (2015) Isolation and biochemical characterization of Apios tuber lectin. Molecules 20:987–1002. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / molecules20010987 22. Soga K, Abo H, Qin SY et al (2015) Mammalian cell surface display as a novel method for developing engineered lectins with novel characteristics. Biomol Ther 5:1540–1562. https://doi.org/10.3390/biom5031540 23. Shimokawa M, Nsimba-Lubaki SM, Hayashi N et al (2014) Two jacalin-related lectins from seeds of the African breadfruit (Treculia africana L.). Biosci Biotechnol Biochem 78: 2036–2044. https://doi.org/10.1080/ 09168451.2014.948376 24. Tsutsui S, Dotsuta Y, Ono A et al (2015) A C-type lectin isolated from the skin of Japanese bullhead shark (Heterodontus japonicus) binds a remarkably broad range of sugars and induces blood coagulation. J Biochem 157:345–356. https://doi.org/10.1093/jb/mvu080 25. Shimokawa M, Fukudome A, Yamashita R et al (2012) Characterization and cloning of GNA-like lectin from the mushroom Marasmius oreades. Glycoconj J 29:457–465. https://doi.org/10.1007/s10719-0129401-6

26. Hu D, Tateno H, Kuno A et al (2012) Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose. J Biol Chem 287: 20313–20320. https://doi.org/10.1074/jbc. m112.351965 27. Takahara K, Arita T, Tokieda S et al (2012) Difference in Fine Specificity to Polysaccharides of Candida albicans Mannoprotein between Mouse SIGNR1 and Human DC-SIGN. Infect Immun 80:1699 LP–1706 LP. https://doi.org/10.1128/IAI.06308-11 28. Tateno H, Toyota M, Saito S et al (2011) Glycome diagnosis of human induced pluripotent stem cells using lectin microarray. J Biol Chem 286:20345–20353. https://doi.org/ 10.1074/jbc.M111.231274 29. Yabe R, Tateno H, Hirabayashi J (2010) Frontal affinity chromatography analysis of constructs of DC-SIGN, DC-SIGNR and LSECtin extend evidence for affinity to agalactosylated N-glycans. FEBS J 277:4010–4026. https://doi.org/10.1111/j.1742-4658.2010. 07792.x 30. Tateno H, Ohnishi K, Yabe R et al (2010) Dual specificity of Langerin to sulfated and mannosylated glycans via a single C-type carbohydrate recognition domain. J Biol Chem 285: 6390–6400. https://doi.org/10.1074/jbc. M109.041863 31. Mitsunaga K, Harada-Itadani J, Shikanai T et al (2009) Human C21orf63 is a heparin-binding protein. J Biochem 146:369–373. https://doi. org/10.1093/jb/mvp079 32. Yamasaki S, Matsumoto M, Takeuchi O et al (2009) C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia. Proc Natl Acad Sci U S A 106:1897 LP–1902 L P. h t t p s : // d o i . o r g / 1 0 . 1 0 7 3 / p n a s . 0805177106 33. Takeuchi T, Sennari R, Sugiura K et al (2008) A C-type lectin of Caenorhabditis elegans: its sugar-binding property revealed by glycoconjugate microarray analysis. Biochem Biophys Res Commun 377:303–306. https://doi. org/10.1016/j.bbrc.2008.10.001

Chapter 3 Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content Analyses of Glycan-Binding Proteins Including Natural Anti-Glycan Antibodies Sharad Purohit and Jin-Xiong She Abstract We present here detailed protocols for the newly developed multiplex glycan bead array (MGBA) for the high throughput and high content analyses of various glycan-binding proteins including anti-glycan antibodies. This platform takes advantage of the commercially available Luminex beads to construct glycan arrays that are easily customizable at will and anytime by researchers. The platform allows the simultaneous analyses of up to 500 glycans and 384 samples at a time. By using multiple arrays, a researcher can analyze thousands of glycans and tens of thousands of samples within a short period. The assay is highly sensitive, specific, reproducible, economic, and fast. Furthermore, the bead array platform is approved for use in clinical settings, speeding up the translation of laboratory discoveries into patient care. Key words Carbohydrates, Glycans, Glycan antibodies, Glycoconjugates, Arrays, Luminex, Beads, Polysaccharides, Biomarkers

1

Introduction Cell surface and secretory proteins produced by mammalian cells covalently modified by additions of carbohydrate residues known as glycans after protein translation is completed. These glycans play an essential role in the maintenance of diverse cellular functions in health and diseases. Alterations to glycans, such as increased levels of truncation and branching as well as presence of unusual terminal sequences [1], may cause changes in various physiological and pathogenic processes including oncogenic transformation [2, 3] and autoimmunity [4–6]. In mammals there are a large number of glycan-binding proteins (GBPs) and the three main GBP families are C-type lectins, galectins, and siglecs, which play critical roles in diverse cellular functions such as cell adhesion, signal transduction, and immune response [6]. Other GBPs include proteins involved in mediating intracellular trafficking, bacterial adhesion molecules, bacterial toxins, viral GBPs, and other microbial GBPs, which are

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_3, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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important for pathogen-host interactions. Another main category of GBPs is anti-glycan antibodies [7, 8], which play an essential role in various diseases including autoimmune diseases, cancer, blood transfusion, organ transplants, and responses to vaccines [5, 9– 11]. In addition to blood transfusion, anti-glycan antibodies may also have a tremendous potential as diagnostic and prognostic markers for other diseases such as cancer and autoimmunity [5, 9–11]. Unlike proteins, glycans do not follow a template for their synthesis; hence, measurement of glycans and their interaction with GBPs is difficult. The advent of the solid surface glycan array developed by the consortium of functional glycomics (CFG) partially alleviated this obstacle [12, 13]. The glycan array approach had been instrumental in greatly enhancing our understanding of glycan functions and their interactions with GBPs [13, 14]. For example, using the planar glycan array the presence of antibodies in human serum to a variety of simple sugars and structurally complex glycans was reported [7, 8]. However, the technical and instrumental challenges associated with solid glycan array make its availability difficult to most biologists. It is almost impossible to translate solid microarray into a clinical tool due to the technical difficulties and long turn-around time of the assay. Furthermore, the solid array also does not allow rapid analyses of large numbers of samples required by many studies. The ability to probe the interaction between glycans and GBPs is of critical importance to biomedical research and clinical care of patients. Therefore, there is still an urgent need for improved and affordable technologies that can analyze large numbers of samples and large numbers of glycans simultaneously. Development of such a high throughput and high content platform that is useful to all biologists and clinic technologists will greatly speed up glycomic studies and the translation of glycomic discoveries into clinical tests. The Luminex bead array consists of 5.6 μm fluorescent microspheres (Fig. 1), each distinguished by a different mixture of red and orange dye. The microspheres are excited with a red laser, and the resultant emission wavelengths can distinguish 500 unique spectrally encoded regions [15, 16]. The high throughput, high content technology has been widely used to analyze plasma proteomics [17], autoimmunity [18], kinase inhibitor [19], protein phosphorylation [20], miRNA [21] and gene expression [22]. One research group has previously attempted to use the Luminex beads for glycan studies [23–25]. However, these studies used a cumbersome conjugation procedure and only a few glycans and a small number of serum samples were analyzed in their studies, which do not allow a rigorous evaluation of the specificity, sensitivity, and reproducibility of their assays. We developed a simple, efficient and reproducible one-step procedure to conjugate glycans and presented our first version of the Multiplex Glycan Bead Array

Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content. . . Create array and analyze sample

Conjugaon

Analysis

L2103

L2103

60

H0403

H0403

OG104 OG104

H0403

H0403

5

H0403

L2103

OG104

0

L2103

MFI (x1000) 10 15 20

25

L2103

35

OG104

OG104

Fig. 1 Multiplex glycan bead array (MGBA) and workflow for high throughput and high content analyses of glycan-binding proteins

(MGBA) that can simultaneously analyze the interaction between hundreds of glycans and various types of GBPs including naturally occurring anti-glycan antibodies [26, 27]. Using a variety of glycan-binding proteins including 39 plant lectins, 13 recombinant anti-glycan antibodies, 4 mammalian GBPs and close to 1000 serum samples, we demonstrated that MGBA was both high content and high throughput and therefore were suitable for the analyses of large numbers of biological samples and glycans [26, 27]. We validated the specificity of the binding on MGBA using three different types of proteins that can bind to glycans: plant lectins, anti-glycan antibodies, and mammalian glycanbinding proteins (selectins and galectins) [26, 27]. For almost all plant lectins, the binding characteristics are largely consistent with the CFG data and previous knowledge. This technology has achieved the sensitivity to recognize the profiles of human anti-glycan antibodies with as little as a few microliters of serum specimen [26, 27]. By creating multiple panels, the bead array has endless capacity to cover the entire glyco-space. The technology can be used not only for biomedical discoveries but also amenable to clinical tests.

2 2.1

Materials Buffers

1. Phosphate buffered saline: 50 mM sodium phosphate pH 7.4 containing 150 mM NaCl. 2. 25 mM Tris–HCl buffer, pH 7.8 containing 75 mM NaCl. 3. Wash buffer 1: PBS containing 0.05% (v/v) Tween-20. 4. Wash buffer 2: Tris–HCl Buffer containing 0.025% (v/v) Tween-20.

2.2

Blocking Buffers

1. Blocking buffer 1: Prepare 1% (w/v) bovine serum albumin (BSA) in PBS. Add sodium azide to 0.2% final concentration.

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Sharad Purohit and Jin-Xiong She

Let it stand at room temperature for 72 h. Filter the solution using a 0.22 μm filter and store at 4
C. 2. Blocking buffer 2: Prepare 1% (w/v) human serum albumin in PBS. Add sodium azide to 0.2% final concentration. Let it stand at room temperature for 72 h. Filter the solution using a 0.22 μm filter and store at 4
C. 3. Blocking buffer 3: Prepare 1% (w/v) BSA in 25 mM Tris–HCl buffer, pH 7.8, containing 75 mM NaCl. Add sodium azide to 0.2% final concentration. Let it stand at room temperature for 72 h. Filter the solution using a 0.22 μm filter and store at 4
C. 4. Blocking buffer 4: Prepare 5% (w/v) BSA in 25 mM Tris–HCl buffer, pH 7.8, containing 75 mM NaCl. Add sodium azide to 0.2% final concentration. Let it stand at room temperature for 72 h. Filter the solution using a 0.22 μm filter and store at 4
C. 5. Bead diluent 1: Add 1% PEG 3000 to blocking buffer 1. 6. Bead diluent 2: Add 0.8% polyvinyl pyrrolidone (MW 360,000) and 1% PEG 3000 to blocking buffer 3. 7. Bead diluent 3: Add 1% PEG 3000 to blocking buffer 2. 2.3 Carboxylated Microspheres and Instrument

1. Microplex carboxylated microspheres from Luminex Corp: One hundred spectrally distinct color coded polystyrene micro-particles from Luminex Corp Cat#LC10XXX-YY, where XXX is three-digit spectral region/identity of the microspheres. YY is the volume/vial size. Dye-encoded beads are created by a precise ratio of red and infrared fluorescent dyes inside the beads, which can be identified by emission wavelength in a Flex-MAP3D (FM3D) or LX-200 analyzer. 2. FM3D or LX200 instrument from Luminex Corp. 3. Rotary shaker with plate holder from IKA MTS4, Wilmington, NC, USA. 4. Vacuum manifold 96-well from Millipore. 5. Multiscreen 384-well plate with polyvinylidene fluoride (PVDF) membrane from Millipore.

2.4 Conjugation Reagents

1. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Pierce cat. no. 22980: Prepare a fresh solution at 10 mg/ml in water immediately before use. EDC is highly hygroscopic. Weigh required amount of EDC into individual tubes on the day of conjugation. Discard EDC solution after every eighth tube. 2. Deionized water freshly taken from the deionizer just before the start of conjugation process.

Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content. . .

2.5 Lectins, Anti-Glycan Antibodies, and Glycan-Binding Proteins

37

1. Biotinylated lectins. The binding of thirty-nine plant lectins (Vector Laboratories) was tested on the glycan array (Table 1). All lectins except AAL should be diluted to working concentration of 5 μg/ml with 1% BSA in PBS (blocking buffer 1) freshly prepared on the day of the assay. Dilute AAL to a concentration of 0.5 μg/ml in blocking buffer 1. 2. Monoclonal anti-glycan antibodies. We used thirteen antiglycan antibodies obtained from commercial sources or produced in-house for characterization of glycan array (Table 2). 3. Glycan-binding proteins (E-selectin, galectin-3, siglec) with Fc tag were from Biolegend. 4. Streptavidin R-phycoerythrin was from One Lambda (Thermo Scientific).

2.6 Free Amine Group Containing Glycans

1. Glycans with 3- or 4-carbon spacer terminating into free amino (–NH2) group were synthesized using Core Synthesis and Enzymatic Extension approaches [28, 29]. 2. Natural glycans purified from several sources were labeled with bifunctional fluorescent tag 2-amino(N-aminoethyl) benzamide (AEAB) [30].

3

Methods

3.1 Conjugation of Glycans to Microbeads

1. Label 1.5 ml micro-centrifuge tube with name of glycan and the bead region before the start of conjugation. 2. On the day of conjugation, adjust the vacuum aspirator to minimum level to aspirate the liquid gently. 3. Vortex the stock vial of beads (1.25  107 beads/ml) vigorously for 30 s, and pipette 100 μl (1.25  106 beads) into a 1.5 ml polypropylene tube. Let the beads settle down for 30 min. 4. Centrifuge the tubes at 12,000  g for 8 min at room temperature to pellet the beads. Aspirate the storage solution by gentle vacuum. 5. Add 100 μl deionized water to the beads, without disturbing the bead pellet using a gel-loading tip. Centrifuge and remove the water as mentioned in step 4. 6. Resuspend the beads in 80 μl of deionized water, to which add 2.5 μg of glycan (1 mg/ml). 7. Add 2.5 μl of EDC solution (10 mg/ml in deionized water) to initiate conjugation. 8. Incubate the beads on a shaker at room temperature for 2 h. After every 30 min, add 2.5 μl of EDC solution (10 mg/ml in deionized water) to the conjugation mixture. Repeat one more time for a total three additions of EDC.

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Sharad Purohit and Jin-Xiong She

Table 1 List of lectins obtained from Vector Laboratories which were analyzed for glycan binding Lectin

Acronym

Sugar Specificity

Ricinus communis II

RCAII

Gal, GalNAc

Erythrina cristagalli

ECL

Gal-β-(1,4)-GlcNAc

Succinylated wheat germ

SuWGA

GlcNAc

Lens clunaris

LCA

α-Man,α-Glc

Ricinus communis I

RCAI

Gal

Jacalin

Jacalin

Gal-β-(1,3)-GalNAc

Pisum sativum

PSA

α-Man,α-Glc

Succinylated Concanavalin A

SuConA

α-Man,α-Glc

Soyabean

SBA

α > β-GalNAc

Dolichos biflorus

DBA

α-GalNAc

Galanthus nivalis

GNL

α-Man

Psophocarpus tetragonolobus I

PTLI

GalNAc, Gal

Phaseolus vulgaris E

PHA-E

Lycopersicon esculentum

LEL

(GlcNAc)2-4

Maackia amurensis I

MALI

Gal-β-(1,4)-GlcNAc

Vicia villosa

VVL

GalNAc

Solanum tuberosum

STL

(GlcNAc)2-4

Phaseolus vulgaris L

PHA-L

Amaranthus caudatus

ACL

Gal-β-(1,3)-GalNAc

Peanut

PNA

Gal-β-(1,3)-GalNAc

Ulex europaeus

UEA

α-Fuc

Narcissus pseudonarcissus

NPL

α-Man

Euonymus europaeus

EEL

Gal-α-(1,3)-Gal

Griffonia simplicifolia II

GSLII

α- or β-GlcNAc

Lotus tetragonolobus

LTL

α-Fuc

Sambucus nigra

SNA

Neu5Ac-α-(2,6)-Gal/GalNAc

Bauhinia purpurea

BPL

Gal-β-(1,3)-GalNAc

Hippeastrum hybrid

HHL

α-Man

Musa Paradisiaca

MPL

α-Man, α-Glc

Sophora japonica

SJA

β-GalNAc

Datura Stramonium

DSL

(GlcNAc)2-4

Wisteria floribunda

WFL

GalNAc

Griffonia simplicifolia I

GSLI

α-Gal, α-GalNAc (continued)

Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content. . .

39

Table 1 (continued) Lectin

Acronym

Sugar Specificity

Concanavalin A

ConA

α-Man, α-Glc

Psophocarpus tetragonolobus II

PTLII

GalNAc, Gal

Griffonia simplicifolia I isolectin B4

BSIB4

α-Gal

Maackia amurensis II

MALII

Neu5Ac-α-(2,3)-Gal-β-(1,3)-GalNAc

Aleuria aurantia

AAL

Fuc-α-(1,6)-GlcNac

Wheat germ

WGA

GlcNAc

Table 2 Anti-glycan antibodies produced in mice/rat used for identifying glycan epitopes. All antibodies were used at a dilution of 1:100 in 1% BSA in PBS (w/v). Muc1, Rat IgG, and mouse anti-goat antibodies were used as negative controls Anti-glycan antibody

Company

CA19.9 Le

Cat #

Ig Type

ABCAM

ab3982

IgM

x

ABCAM

ab3358

IgM

y

ABCAM

ab3359

IgM

Le IgM SSEA3 GM1 Gal α1,3 Sle

x

GA1 b

a

MC631

IgM

a

D0131

IgM

In lab In lab

a

IgM

In lab

a

SNH3

IgM

a

D079

IgM

In lab In lab

ABCAM

ab3968

IgG

Blood group H2

ABCAM

ab33404

IgG

CA19.9

US Bio

C0075-31

IgG

ab3967

IgG

Le

Le

a Y

a

Clone ID

5G17

ABCAM a

Le IgG

In lab

E2014

Muc1

Fitzgerald

M3A106

Muc1

BBI solutions

Mouse anti-goat IgG HRP

Pierce

Rat IgG

Pierce

Polyclonal

IgG 10-R-M129C

IgG

BM236-V2G9

IgG IgG IgG

These monoclonal antibodies were produced in laboratory by immunizing intact cells into mice. Individual clones were identified by inhibition studies (unpublished results)

40

Sharad Purohit and Jin-Xiong She

9. Incubate the reaction mixture for 2 h and then add 20 μl of 200 mM solution of disodium hydrogen phosphate in water to beads and incubate for 30 min. This is the first stop you can leave the beads at this step for overnight at 4
C. 10. Add 2 μl of 0.1% (v/v) aqueous Tween-20 solution to the tubes, vortex to mix, and let the mixture stand for 15 min at room temperature. Centrifuge at 12,000  g for 8 min and remove supernatant by vacuum aspiration. 11. Block beads recovered from above step with 1 ml of blocking buffer 1 for 24–72 h at room temperature with end-to-end shaking. 12. After blocking, collect glycan-conjugated beads by centrifugation, resuspend in PBS containing 1% BSA (w/v) and 0.2% sodium azide (w/v), and store in the dark at 4
C. For measurement of human IgG, use blocking buffer 2 to block the beads. 13. To assess the assay background, beads from three different bead regions were included as “no glycan conjugation” (NGC) controls. These NGC control beads underwent the same conjugation process at the same time as the glycanconjugated beads. 3.2 Characterization of Glycan-Binding Epitopes Using Lectins

1. Before the assay, wash the wells of a 384-well filter plate (EMD Millipore, MS, USA), by filling all individual wells with 90 μl wash buffer 1. Remove the wash buffer by applying vacuum suction. 2. Create a working glycan array by mixing individual glycan beads in a tube. Dilute the working bead array to a final dilution of 1:100 with bead diluent 1. Remember to add the NGC control beads to the bead mixture for background control at same dilution. 3. Using a multi-channel micropipette, add 10 μl of the microsphere suspension to each well, followed by addition of 10 μl of diluted biotinylated lectin solutions. 4. Incubate the lectin and bead mixture at room temperature on a shaker set at 550 rpm for 1 h. 5. Remove unreacted lectin solution by vacuum suction and wash the wells twice with wash buffer 1. To wash, fill the wells with 75 μl of wash buffer 1. Remove the buffer by applying vacuum suction. 6. Add 10 μl of streptavidin R-phycoerythrin (SAPE; 3 μg/ml freshly diluted in wash buffer 1). Incubate the plate with SAPE for 30 min at room temperature on a shaker. 7. Remove all liquid from the wells with vacuum suction and wash two times with wash buffer 1.

Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content. . .

41

8. Resuspend beads in 60 μl of wash buffer and read fluorescence intensities (MFI) on a FlexMAP 3D reader (Millipore, Billerica, MA) with the following instrument settings: events/bead: 50, minimum events: 0, flow rate: 60 μl/min, sample size: 50 μl, and doublet discriminator gate: 8000–13,500. 3.3 Analyses of Monoclonal and Polyclonal Anti-Glycan Antibodies

1. Mix individual beads together and dilute (1:100) in bead diluent 1 to prepare the glycan array. 2. Dilute antibodies to 2 μg/ml in blocking buffer 1. 3. Wash required number of wells of a 384-well plate with 90 μl of wash buffer 1. 4. Add 10 μl of diluted beads to prewashed wells of a 384-well filter plate followed by 10 μl of diluted antibodies. 5. Incubate the mixture at room temperature for 1 h on a shaker set at 550 rpm. After the incubation time is over, separate antiglycan antibody bound beads from reaction mixture by vacuum suction. 6. Remove unbound antibodies by washing the wells with wash buffer 1. To wash, add 75 μl of wash buffer 1 to the wells, and then remove it by vacuum suction. Repeat the procedure one more time. 7. Add PE-labeled anti-mouse IgM or anti-mouse IgG (2.5 μg/ ml diluted in blocking buffer 1; Southern Biotech, AL, USA) to the wells and incubate for one additional hour with shaking. 8. Remove the unbound secondary antibody by vacuum suction and wash wells with wash buffer 1 as described in steps 5 and 6. Resuspend in 60 μl of wash buffer and measure MFI on FM3D reader with settings described in Subheading 3.2.

3.4 Glycan-Binding Protein (GBP) Assay

1. GBPs were multimerized according to Blixt et al. [31]. Briefly, mix GBP-Fc solution (25 μg/ml in blocking buffer 3 containing 1 μg/ml of rat IgG) with secondary and PE-labeled tertiary antibody in a ratio of 1:0.5:0.25 and incubate at 37
C for 30 min to form multivalent GBP-antibody complexes. 2. Mix individual glycan beads and dilute 1:100 in bead diluent 2 to get a final bead count of 1000 beads/glycan. Pipette 10 μl of this bead array into a pre-washed well of 384-well plate. 3. Add 10 μl volume of the GBP-antibody complex mixture to the beads and incubate for 1 h with shaking on a shaker set at 550 rpm. 4. Remove unbound GBP complexes by vacuum suction and wash beads three times with wash buffer 2. To wash, fill the wells with 75 μl of wash buffer 2. Remove the buffer by applying vacuum suction.

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Sharad Purohit and Jin-Xiong She

5. Resuspend the washed beads in 60 μl of wash buffer 2. Read MFI using a Luminex FM3D machine as described in Subheading 3.2, step 8. 3.5 Profiling of Natural Anti-Glycan IgM in Human Serum

1. Dilute human serum samples 2500-fold in blocking buffer 4 containing 0.8% polyvinyl pyrrolidone (MW 3,600,000). 2. Incubate 10 μl of diluted serum sample with glycan bead (1000 beads/glycan prepared in bead diluent 2) for 2 h at room temperature on a shaker set at 550 rpm. 3. After 2 h, remove unbound reagents by vacuum suction and wash the wells with wash buffer 2. 4. Add diluted biotinylated anti-human IgM (3 μg/ml in blocking buffer 3; Southern Biotech, AL, USA) to each well and incubate the plate for 1 h. 5. Remove unbound reactants by vacuum suction. Wash the plate with 75 μl of wash buffer 2. To wash, fill the wells with 75 μl of wash buffer 2. Remove the buffer by applying vacuum suction. 6. Dilute stock SAPE in wash buffer 2 to final concentration of 3 μg/ml. Add 10 μl of diluted SAPE to individual wells and incubate the plate for 30 min at room temperature with shaking (550 rpm). 7. Wash the plate two times with 75 μl of wash buffer 2 to remove the unbound SAPE as described in step 5 of this section. 8. Resuspend beads in 60 μl of wash buffer 2. Read MFI using Luminex FM3D machine using settings described in Subheading 3.2.

3.6 Measurement of Natural Anti-Glycan IgG Antibodies in Human Serum

1. Dilute human serum samples 500-fold in blocking buffer 1. 2. Incubate 10 μl of diluted serum sample with 1000 microspheres for each glycan (diluted in bead diluent 3) for 2 h at room temperature on a shaker set at 550 rpm. 3. Remove unbound reagents by vacuum suction and wash the wells with wash buffer 1. To wash, fill the wells with 75 μl of wash buffer 1. Remove the buffer by applying vacuum suction. 4. Add biotinylated anti-human IgG (3 μg/ml in blocking buffer 2; Southern Biotech, AL, USA) to each well and incubate the plate for 1 h on a shaker at room temperature. 5. Remove the unbound secondary antibody by washing the plates two times with wash buffer 1, described in step 3 above. 6. Prepare SAPE (3 μg/ml solution) in wash buffer 1, add 10 μl of it to each well and incubate for 30 min at room temperature on a shaker (550 rpm).

Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content. . .

43

7. Wash the plate two times with wash buffer 1 and resuspend beads in 60 μl of wash buffer. Read MFI using Luminex FM3D machine with settings described in Subheading 3.2.

Acknowledgments This work was partially supported by National Institute of Health (NIH)/National Cancer Institute (NCI) grants 1 R21 CA199868 and U01CA221242. References 1. Kim YJ, Varki A (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj J 14:569–576 2. Amano J, Nishimura R, Sato S, Kobata A (1990) Altered glycosylation of human chorionic gonadotropin decreases its hormonal activity as determined by cyclic-adenosine 30 ,50 -monophosphate production in MA-10 cells. Glycobiology 1:45–50 3. Powlesland AS et al (2009) Targeted glycoproteomic identification of cancer cell glycosylation. Glycobiology 19:899–909 4. Marth JD, Grewal PK (2008) Mammalian glycosylation in immunity. Nat Rev Immunol 8: 874–887 5. Arnold JN, Saldova R, Hamid UM, Rudd PM (2008) Evaluation of the serum N-linked glycome for the diagnosis of cancer and chronic inflammation. Proteomics 8:3284–3293 6. Varki A, Lowe JB (2009) Biological roles of Glycans. In: Varki A et al (eds) Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY) 7. Oyelaran O, McShane LM, Dodd L, Gildersleeve JC (2009) Profiling human serum antibodies with a carbohydrate antigen microarray. J Proteome Res 8:4301–4310 8. Bovin N et al (2012) Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies? Biochim Biophys Acta 1820:1373–1382 9. Campbell CT et al (2014) Humoral response to a viral glycan correlates with survival on PROSTVAC-VF. Proc Natl Acad Sci U S A 111:E1749–E1758 10. Kaul A et al (2012) Serum anti-glycan antibody biomarkers for inflammatory bowel disease diagnosis and progression: a systematic review and meta-analysis. Inflamm Bowel Dis 18: 1872–1884 11. Saldova R et al (2007) Ovarian cancer is associated with changes in glycosylation in both

acute-phase proteins and IgG. Glycobiology 17:1344–1356 12. Song X et al (2011) Shotgun glycomics: a microarray strategy for functional glycomics. Nat Methods 8:85–90 13. Song X et al (2009) Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem Biol 16:36–47 14. Yu Y et al (2014) Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses. Mol Cell Proteomics 13: 2944–2960 15. Dunbar SA (2013) Bead based suspension arrays for the detection and Identificatin of respiratory viruses. In: Tang Y-W, Stratton CW (eds) Advanced techniques in diagnostic microbiology. Springer, New York, pp 813–833 16. McDonald JU, Ekeruche-Makinde J, Ho MM, Tregoning JS, Ashiru O (2016) Development of a custom pentaplex sandwich immunoassay using protein-G coupled beads for the Luminex(R) xMAP(R) platform. J Immunol Methods 433:6–16 17. Bystrom S et al (2014) Affinity proteomic profiling of plasma, cerebrospinal fluid, and brain tissue within multiple sclerosis. J Proteome Res 13:4607–4619 18. Ayoglu B et al (2016) Anoctamin 2 identified as an autoimmune target in multiple sclerosis. Proc Natl Acad Sci U S A 113:2188–2193 19. Muellner MK et al (2011) A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer. Nat Chem Biol 7: 787–793 20. Du J et al (2009) Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy. Nat Biotechnol 27:77–83 21. Lu J et al (2005) MicroRNA expression profiles classify human cancers. Nature 435:834–838

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22. Peck D et al (2006) A method for highthroughput gene expression signature analysis. Genome Biol 7:R61 23. Pochechueva T et al (2014) PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array. J Immunol Methods 412:42–52 24. Pochechueva T et al (2011) Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies. Glycoconj J 28:507–517 25. Pochechueva T et al (2011) Multiplex suspension array for human anti-carbohydrate antibody profiling. Analyst 136:560–569 26. Purohit S et al (2018) Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins. Nat Commun 9:258 27. Purohit S et al (2020) Better survival is observed in cervical cancer patients positive for specific anti-glycan antibodies and receiving

brachytherapy. Gynecol Oncol 157(1): 181–187 28. Xia L, Gildersleeve JC (2015) The glycan Array platform as a tool to identify carbohydrate antigens. In: Lepenies B (ed) Carbohydratebased vaccines: methods and protocols. Springer, New York, New York, NY, pp 27–40 29. Bohorov O, Andersson-Sand H, Hoffmann J, Blixt O (2006) Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans. Glycobiology 16:21c–27c 30. Song X, Lasanajak Y, Xia B, Smith DF, Cummings RD (2009) Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem Biol 4:741–750 31. Blixt O et al (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci U S A 101: 17033–17038

Chapter 4 Nanocube-Based Fluidic Glycan Array Hung-Jen Wu, Akshi Singla, and Joshua D. Weatherston Abstract The nature of cell membrane fluidity permits glycans, which are attached to membrane proteins and lipids, to freely diffuse on cell surfaces. Through such two-dimensional motion, some weakly binding glycans can participate in lectin binding processes, eventually changing lectin binding behaviors. This chapter discusses a plasmonic nanocube sensor that allows users to detect lectin binding kinetics in a cell membrane mimicking environment. This assay only requires standard laboratory spectrometers, including microplate readers. We describe the basics of the technology in detail, including sensor fabrication, sensor calibration, data processing, a general protocol for detecting lectin-glycan interactions, and a troubleshooting guide. Key words Nanocube sensor, Lectin, Supported lipid bilayer, Glycan array, Multivalent binding, Hetero-multivalency, Fluidic membrane, Localized surface plasmon resonance, Reduction of dimensionality

1

Introduction Most lectins bind to glycans via multivalent interactions to enhance the overall binding avidity [1]. Because of the fluidic nature of cell membranes, glycans attached to lipids and proteins on cell membranes can freely diffuse in two dimensions. Such two-dimensional motion allows glycans to organize their positions via random diffusion, and eventually lead to multivalent interactions with lectins. The 2D motion of glycans can significantly alter the reaction rate between a glycan and a lectin [2]. Figure 1 illustrates how a pentavalent lectin, cholera toxin subunit B (CTB), binds to glycolipids on cell membranes. After a CTB attaches to the first glycolipid, both bound CTBs and unbound glycolipids can freely diffuse and collide on the 2D cell membrane. Due to the reduced dimension of diffusion, the reaction rates of subsequent bindings on 2D cell membrane surfaces are at least 104 times higher than the first binding, which occurred in a three-dimensional (3D) space [2]. This intrinsic rate enhancement mechanism enables the low-affinity ligands, whose dissociation constants are in the

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_4, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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Fig. 1 (a) Hetero-multivalent binding mechanism. Homo-oligomeric pentavalent lectins (e.g., cholera toxin subunit B, CTB) diffuse from the solution phase to cell membrane surfaces and bind to a high-affinity ligand. Free glycolipid ligands move two dimensionally, encounter bound lectins, and enable subsequent binding. Due to the reduced dimension of diffusion, the reaction rates of subsequent bindings on 2D membrane surfaces are at least 104 times higher than the first binding in 3D space. (b–d) Hetero-multivalency on a membrane surface (top-down view) (b) A membrane surface with low-affinity ligands only (e.g., GM2 for CTB, dissociation constant, Kd > 1 mM). Because the affinity is extremely low, lectins cannot bind to the surface at physiological concentrations. (c) A membrane surface with only high-affinity ligands (e.g., GM1 for CTB, Kd
10 nM). Because the subsequent binding rate significantly increases, a bound lectin rapidly binds to four other ligands. No more free ligand is available to accept additional lectins. (d) A membrane with both high- and low-affinity ligands. Due to rate enhancement, even low-affinity ligands can now participate in the second or higher binding events. If the density of low-affinity ligand is sufficient, the low-affinity ligands compete with highaffinity ligands in lectin binding. Thus, some high-affinity ligands become available to accept more lectins from the solution phase, leading to an increased number of bound lectins. Although the number of bound lectins (i.e., binding capacity) increases, the binding energy per lectin (i.e., binding avidity) decreases

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millimolar range, to participate in the subsequent binding events even though the concentration of lectin is in the nanomolar range. This hetero-multivalent binding process increases the total number of available binding sites on the cell surface, leading to an increased number of bound lectins. However, when a single lectin simultaneously binds to high- and low-affinity ligands, the average binding energy per lectin reduces. We recently established a theoretical model to simulate this fundamental phenomenon [3–6]. The computer simulation can directly visualize the time course of the lectin bound states, leading to the discovery of a new ligand exchange mechanism [6]. Occasionally, the high-affinity ligand can dissociate from the bound lectin. If the density of the low-affinity ligand is sufficient, another low-affinity ligand can reach this free binding site before the lectin dissociates from the cell surface. Therefore, a lectin can also be stabilized entirely by low-affinity ligands. By repeating this ligand exchange process, a tiny number of high-affinity ligands can result in a large number of lectin attachments. This ligand exchange mechanism offers a potential answer for a long-standing question, why some lectins could significantly bind to cells when their primary glycan ligands have low abundance [7–19]. The ligand exchange mechanism is highly nonlinear; thus, a small perturbation of cell membrane properties can lead to complex biological outcomes. Our theoretical analysis indicates several essential discoveries [3]. First, the hetero-multivalent binding process has complex impacts on lectin binding capacity and avidity. For example, in Fig. 1, the participation of the low-affinity ligands increased the total number of available binding sites on the cell surface, leading to an increased number of bound CTB (binding capacity). The increase in the binding capacity enhances the probability of endocytosis. However, binding to the low-affinity ligands reduces the average binding energy (avidity) between a lectin and the cell membrane, decreasing the endocytosis rate [20, 21]. Therefore, the balance between binding capacity and avidity determines the intoxication rate of cholera toxins. Second, the hetero-multivalent binding process can significantly alter binding kinetics. The increased density of high-affinity ligands can accelerate the ligand exchange mechanism. In contrast, the binding rates reduce when the density of the low-affinity ligand increases. The time to reach binding equilibrium could vary from a minute to a day [3, 22]. Third, a minimum density is required to trigger the lectin binding to a low-affinity ligand. This threshold density depends on the binding affinity of the lectin with the low-affinity ligand. Fourth, competitive binding of high- and low-affinity ligands (i.e., the number density ratio of high- and low-affinity ligands) could significantly influence the lectin binding process. Some of these theoretical predictions agree with the recent experimental data published by Kohler and her coworkers [22].

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To consider these complex impacts on lectin-glycan interactions in a cell membrane environment, a new detection tool is highly desired. The new tool should include several important features. First, the detection tool should be able to monitor lectin-glycan interactions in an environment which mimics the cell membrane. Second, the tool should be able to measure separately the lectin binding capacity, avidity (dissociation constant), and kinetics. Third, to discover the cooperative actions between multiple glycan ligands in various cell membrane compositions, we need a highly flexible tool that allows users to easily manipulate membrane compositions and observe the lectin binding behaviors in those conditions. Here, we report a nanocube-based fluidic glycan array tool to address the abovementioned challenges [23, 24] (Fig. 2). A plasmonic sensor, silver-silica core-shell (Ag@SiO2) nanocube, is used to monitor lectin-glycan interactions in a cell membrane environment. Supported lipid bilayers (SLBs) form spontaneously on sensor surfaces upon mixing Ag@SiO2 nanocubes with a liposome suspension. A thin water layer (~1 nm) resides between the SLBs and the sensor surfaces so that the lipid bilayer can mimic the cell membrane and possesses the same two-dimensional fluidity. Lectin binding to glycans in the model membrane is monitored by observing the transmission spectrum shift of the localized surface plasmon resonance (LSPR). This label-free detection tool does not require fluorescent probes or antibodies to label the analytes, and thus, it can monitor lectin-glycan interactions in their native states. Additionally, in contrast to a fluorescence-based approach that suffers photobleaching problems, LSPR detection can monitor lectin binding kinetics at small time intervals. Moreover, this sensing platform does not require daily calibrations for absolute quantification of lectin binding to glycans; thus, it is an ideal tool to quantify the absolute bound lectin density (i.e., binding capacity). Because LSPR detection only requires the measurement of transmission spectra, users can use any common ultraviolet–visible (UV-Vis) spectrometers, including microplate readers, without purchasing new instruments. High-throughput utility can be achieved by using microplate readers equipped with automated reading, injection, and shaking accessories. In this chapter, we first provide the nanocube synthesis protocol and sensor calibration procedure. The fabrication of liposomes containing glycolipids and the formation of SLBs are described as well. We also discuss data processing and include a troubleshooting guide.

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Fig. 2 Workflow of the nanocube-based assay

2

Materials

2.1 Synthesis of Plasmonic Silver Nanocube Sensor

1. Chemicals: Copper(II) chloride dihydrate (CuCl2, SigmaAldrich, Cat. #: 307483), polyvinylpyrrolidone (PVP, molecular weight ¼ 55,000, Sigma-Aldrich, cat. no. 856568), silicone oil (working temperature: 50  C to +200  C, Sigma-Aldrich, cat. no. 85409), silver nitrate (AgNO3, 99.9995%, 100 g, Alfa Aesar, Cat. #: 10858), 1,5-pentanediol (PD, 98%, Acros Organics, Cat#: AC12995-0010), ethanol (200 proof, Sigma-Aldrich, cat. no. EX0276), hydrochloric acid (HCl, certified ACS Plus, 36.5 to 38.0%, Fisher Chemical, cat. no. A144S-500), nitric acid (HNO3, certified ACS Plus, 68.0% to 70.0%, Fisher Chemical, cat. no. A200C-212), isopropyl alcohol (IPA, certified ACS Reagent, >99.5%, Fisher Chemical, cat. no. A416-4), sodium hydroxide (NaOH, pellets, Alfa Aesar, cat. no. A16037). 2. Base bath for cleaning glassware: approximately 200 g of NaOH added to 4 L of IPA and 1 L of deionized (DI) water. The base-bath solution should be stored in a plastic bucket with a secondary container. Caution: The base-bath solution is corrosive. The preparation and use of base bath requires appropriate personal protective equipment, including lab coat, closetoed shoes, safety goggles, and heavyweight gloves. 3. Aqua regia for removing metal contaminants on the glassware: 4 parts (volume) of concentrated HCl mixed with 1 part of concentrated HNO3 to reach 3:1 molar ratio. Caution: Aqua regia is highly corrosive and generates toxic vapors containing NO2 and Cl2. The preparation and use of aqua regia should be conducted in a fume hood with appropriate personal protective

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equipment, including lab coat, rubber apron, close-toed shoes, safety goggles, face shield, and heavyweight gloves. 4. Synthesis solutions: The following chemicals have to be dissolved in highly viscous 1,5-pentanediol (PD) (see Notes 1 and 2). Using bath sonication and vortex mixing repeatedly can help dissolve the chemicals in PD. CuCl2 solution: 0.082 g of CuCl2 in 10 mL of PD. PVP solution: 0.2 g of PVP in 10 mL of PD. AgNO3 solution: 0.2 g of AgNO3 in 10 mL of PD. Add 30 μL of CuCl2 solution into the AgNO3 solution 20 min before reaction. 5. Membrane filter for nanocube purification: Millipore filter membranes, Durapore® PVDF (diameter: 47 mm, pore size: 5 μm (cat. no. P8949), 0.45 μm (cat. no. P1938), and 0.22 μm (cat. no. P1438)). 2.2 Synthesis of Silver-Silica CoreShell (Ag@SiO2) Nanocube Sensor

1. Chemicals: Isopropyl alcohol (IPA, certified ACS Reagent, >99.5%, Fisher Chemical, cat. no. A416-4), tetraethyl orthosilicate (TEOS, Sigma-Aldrich, cat. no. 86578), Ammonium hydroxide solution (28–30%, ACS Reagent, Sigma-Aldrich, cat. no. 320145).

2.3 Preparation of Lipid Bilayer Coated Ag@SiO2 Nanocubes

1. Chemicals: Chloroform (ACS Reagent, Spectroscopy Grade, 99.8%, Acros Organics, cat. no. 404635000), 1 phosphatebuffered saline (PBS, 10 concentrate, BioUltra, SigmaAldrich, cat. no. 79383). 2. Lipids: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N(cap biotinyl) (biotinyl-cap PE, Avanti Polar Lipids, Inc., cat. no. 870273C), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids, Inc., cat. no. 850375C), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS, Avanti Polar Lipids, Inc., cat. no. 840035C). 3. 0.05 g/L of bovine serum albumin (BSA, Sigma-Aldrich, cat. no. A2153) is prepared in 1X PBS for blocking non-specific adsorption.

2.4 Ag@SiO2 Nanocube Sensor Calibration

1. Chemicals: Glycerol (99.5%, Sigma-Aldrich, cat. no. G9012), streptavidin (Sigma-Aldrich, cat. no. 189730), 1 phosphatebuffered saline (PBS, 10 concentrate, BioUltra, SigmaAldrich, Cat. #: 79383). 2. 0.05 g/L of bovine serum albumin (BSA, Sigma-Aldrich, cat. no. A2153) is prepared in 1 PBS for blocking non-specific adsorption.

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1. Lectins, biological samples (e.g., serum, culture media), and cells (e.g., bacteria) of interest: The localized surface plasmon resonance (LSPR) detection is a label-free detection method. There is no need of antibodies or labeling reagents. The detection can be conducted in the native states of biological samples. However, the samples should not contain reducing reagents that are often used to stabilize cysteine residues. 2. Glycolipids and neoglycolipids: Glycolipids and neoglycolipids can be purchased from Matreya LLC (State College, Pennsylvania, USA), OligoTech (Crolles, France), and Dextra Laboratories Ltd. (Reading, UK). 3. Lipids: A wide range of cell membrane molecules, including phospholipids, sphingolipids, and cholesterols, can be purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA) and Sigma-Aldrich.

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Methods

3.1 Synthesis of Plasmonic Nanocube Sensor 3.1.1 Synthesis of Nanocubes

1. Clean all glassware with base bath before synthesis. Immerse glassware in base bath for at least 4 h and then rinse with double deionized (DDI) water. Dry the glassware. 2. Heat the silicone oil bath to 190  C using a hot plate with precise temperature control (e.g., Ika RET control-visc connecting a contact thermometer). Turn the magnetic stir to 300 rpm. To reach steady state, heat the bath for at least 1 h before the reaction. 3. Before synthesis, clean the reactor (KONTES Round-Bottom Boiling Flask,100 mL) with aqua regia for 15 min followed by rinsing with DDI water at least 20 times and methanol five times (see Note 3). 4. Pour 20 mL of PD in the reactor. Immerse the reactor into the oil bath. Heat the reactor and measure the temperature inside with a thermometer. 5. When the temperature of PD inside the reactor reaches 130  C (see Note 4), add 250 μL of AgNO3 solution, quickly followed with 500 μL of PVP solution. 6. After 35 s, add 500 μL of AgNO3 solution, and quickly followed with 500 μL of PVP solution. 7. Wait 60 s, then add 500 μL of AgNO3 solution, quickly followed by 500 μL of PVP solution. 8. Continue step 7 until all the 10 mL of AgNO3 and PVP solutions have been added. 9. Wait 1 min, then remove the reactor from the silicone oil bath and let the solution cool to room temperature.

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10. Pour the solution into two 50 mL falcon centrifuge tubes. Add ethanol to fill the centrifuge tubes up to 50 mL. 11. Centrifuge the tubes at 3260  g for 30 min. Remove the supernatant. Add 20 mL of 200 proof ethanol. Use sonication to re-suspend particles. Combine the contents of the two tubes into a single 50 mL tube. 12. Fill the tube with ethanol. Centrifuge at 3260  g for 30 min. Remove the supernatant and fill the tube with 200 proof ethanol. Use sonication to re-suspend particles. 13. Repeat step 12 three times to remove the reactants. Store the nanocubes in 50 mL of ethanol. 3.1.2 Nanocube Purification

1. Prepare PVP aqueous solution (add 2 g of PVP in 300 mL DDI water). Mix 50 mL nanocubes in ethanol with 50 mL of PVP aqueous solution in a beaker. 2. Use Sigma-Aldrich vacuum filtration system (cat. no. Z290432) with the PVDF membrane filters (see Subheading 2.1) to purify nanocube particles. Pour the nanocube + PVP solution through the filtration setup. After all particles go through the filter, rinse the funnel with 200 proof ethanol. Keep the filtrate and discard the filter. Change the filter for the next filtration. 3. Start with 5 μm pore size PVDF filter, followed by 0.45 μm and finally 0.22 μm pore size filters. This filtration process will remove the rod-shaped particles. To obtain monodisperse nanocubes, filter the particle solution through the 0.22 μm filter at least five times. 4. After filtration, transfer the entire solution to 50-mL falcon centrifuge tubes. Centrifuge the tubes (3260  g for 30 min), remove the supernatant, and re-suspend nanocube particles in ethanol using bath sonication. Repeat this step at least four times. 5. After the last centrifuge, add only 10 mL ethanol in each tube, re-suspend the particles, and combine all the product fractions into a single 50-mL falcon tube. Fill the tube with ethanol up to 50 mL. Cover the tube with aluminum foil to prevent light exposure. The nanocubes remain stable for at least 2 years (see Notes 5 and 6).

3.2 Synthesis of Silver-Silica CoreShell (Ag@SiO2) Nanocube Sensor

All glassware should be cleaned with base bath. 1. Take 20 mL of silver nanocube solution from Subheading 3.1.2 and mix with 30 mL of IPA in a 50 mL falcon tube. Centrifuge at 3260  g for 30 min and remove the supernatant. Fill the tube with 50 mL of IPA. Use sonication to re-suspend the particles.

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2. Repeat the centrifugation three times to re-suspend nanocube particles in IPA. During the last wash, fill the tube with only 20 mL of IPA (see Note 7). 3. Pour the nanocube solution in IPA (20 mL) into a reactor (KONTES round-bottom boiling flasks, 250 mL). Add 55 mL IPA to the reactor. 4. Dilute 120 μL of 28 wt% ammonium hydroxide solution with 3.88 mL of DDI H2O to obtain 0.84 wt% ammonium hydroxide. Standard micropipettes (i.e., air-displacement pipettes) cannot accurately measure the volume of volatile ammonium hydroxide, so piston pipettes should be used. 5. Add 22.1 mL of DDI water to the reactor, followed by 6.80 mL of TEOS, and then by 3.4 mL of 0.84% ammonium hydroxide (ammonium hydroxide acts as a catalyst and hence should be added at the end). 6. Use a magnetic stirrer set to 300 rpm to mix the reactants. Let the solution react for 60 min. 7. After the 60 min reaction, add 50 mL of ethanol to reduce the reaction rate. Pour the solution into 50 mL falcon centrifuge tubes. 8. Centrifuge the particle solution at 3260  g for 30 min. Remove the supernatant. 9. Add 75 mL of IPA, 22.1 mL of DDI water, and 6.80 mL of TEOS in the 50 mL tubes and re-disperse the particles using sonication. Then, transfer the solution back to the 250 mL round-bottom flask reactor. Heat the solution at 60  C in silicone oil bath for 10 h. 10. After 10 h, pour the particle solution into 50 mL falcon tubes. Centrifuge the particle solution at 3260  g for 30 min. 11. Remove the supernatant. Fill each falcon tube with DDI water, and then sonicate the solution for 1 min to re-disperse particles. 12. Centrifuge the particle solution at 3260  g for 10 min. Remove the supernatant, fill the tubes with DDI water, and then re-disperse the particles using sonication. Repeat this washing step four times. 13. After the last washing step, store the silver-silica core-shell (Ag@SiO2) nanocube sensors in 40 mL of DDI water. The sensors remain stable in DDI water for at least 2 years (see Note 8).

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3.3 Preparation of Lipid Bilayer Coated Ag@SiO2 Nanocubes 3.3.1 Preparation of Lipid Solutions

1. Wash a small round-bottom flask (25 mL, PYREX) using base bath. Rinse the flask with DDI water extensively (20 times), followed with methanol. Dry the flask. 2. Rinse the flask twice with chloroform. Mix the stock lipid solutions according to the desired lipid bilayer compositions. For example, to prepare the lipid bilayer for streptavidinbiotinyl lipid calibration (Subheading 3.4.3), mix 265.1 μL DOPC (2.5 mg/mL), 153.5 μL DOPS (0.5 mg/mL), and 209.4 μL biotinyl-capped PE (0.05 mg/mL) in chloroform. This mixture will yield a 250 μL of 3 mg/mL liposome solution with the composition of 89 mol% DOPC, 10 mol% DOPS, and 1 mol% biotinyl-capped PE (see Notes 9 and 10). 3. Use a rotary evaporator to evaporate the organic solvents. Heat the water bath to 40  C to accelerate evaporation. 4. Add 250 μL of DDI water to rehydrate the dried lipids. The freeze-thaw cycle can be used to improve lipid hydration. Shake the lipid solution in the sealed round-bottom flask. Immerse the flask in a dry ice/isopropanol solution to freeze the lipid solution. Then warm the lipid solution in a warm water bath (40  C). Repeat this freeze–thaw cycle three times (see Note 11). 5. The resultant liposome solution contains multilamellar vesicles (MLV). Continue to Subheading 3.3.2 to fabricate small unilamellar vesicles (SUV).

3.3.2 Preparation of SUV Solutions

Extrusion

SUV can be prepared by either extrusion or sonication methods (see Note 12). The extrusion method uses an extruder set (Avanti Polar Lipid, Inc., cat. no. 610000). The sonication method requires a relatively expensive tip ultrasonicator (e.g., Q700, Qsonica, LLC.). The advantage of the sonication method is that it can process multiple samples simultaneously if the ultrasonicator is equipped with a multi-tip horn. 1. Assemble the extruder set (Avanti Polar Lipid, Inc., cat. no. 610000), according to the manual. The filter membrane with 100 nm pore size is used for extrusion. 2. Pass DDI water through the extruder set to ensure that the surface inside the extruder is wet. 3. Pass the MLV lipid solution prepared in Subheading 3.3.1 through the filter 20 times. Store the SUV solution in snapcap polypropylene microcentrifuge tubes at 4  C.

Tip Sonication

1. Keep the MLV lipid solution in a 500 μL snap-cap microcentrifuge tube. To remove the heat generated during sonication, immerse the tube in an ice bath. Use a tube holder or clamp to hold and secure the tube. Do not let the tube float.

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2. Make sure the tip sonicator is clean before use. Switch on the sonicator and set it to the following attributes: amplitude (60%), timer (3 min), pulse (on: 5 s, off: 3 s). 3. After sonication, centrifuge the solution at 13,000  g for 20 min at 4  C to remove any debris resulting from tip sonication. Collect the supernatant in a new 500 μL microcentrifuge tube. 3.3.3 Lipid Bilayer Coating on the Ag@SiO2 Nanocubes

1. Add 500 μL of Ag@SiO2 nanocube solution to a microcentrifuge tube. Centrifuge (5 min at 3260  g) and then remove the supernatant. Add 100 μL of DDI water and re-suspend the particles using sonication. 2. Add 20 μL of the SUV solution to a 500 μL microcentrifuge tube. Then, add 100 μL of Ag@SiO2 solution followed by 120 μL of 2 phosphate-buffered saline (PBS) to the tube. 3. Add 260 μL of 1 PBS to the lipid bilayer coated Ag@SiO2 solution to make the final volume 500 μL. Transfer to a 2 mL Eppendorf tube. Then add 1.5 mL of 1 PBS, bringing the total volume to 2 mL. 4. To block non-specific binding, add 50 μL of 0.05 g/L bovine serum albumin (BSA) solution to the 2 mL of lipid bilayer coated Ag@SiO2 solution and incubate for 1 h, keeping the solution well mixed (do not use sonication after coating lipid bilayer) (see Note 13).

3.4 Sensor Calibration

Within the same synthesis batch, the nanocube sensors provide stable measurements. For glycerol-water calibration (Subheading 3.4.2), the typical inter- and intra-day variation of LSPR locations is less than 0.7% (coefficient of variation, CV). However, the sizes of nanocubes and the thickness of silica shells vary slightly among different synthesis batches, leading to variable detection sensitivity. Thus, in order to compare the binding data measured by different batches of sensors, calibration is required. Users only need to conduct the sensor calibration once when a new batch of sensors is fabricated. Daily calibration is not needed.

3.4.1 Measurement of Localized Surface Plasmon Resonance (LSPR)

LSPR is easily observed by transmission ultraviolet–visible (UV-Vis) spectroscopy. The prominent quadrupolar LSPR peak is determined by fitting a transmission spectrum to a polynomial function. 1. Suspend the nanocube sensors in the desired solution. Use a vortex mixer to disperse the particles. 2. Measure the transmission spectra of the nanocube solutions and the blank solution (i.e., the solution without nanocube sensor) using UV-Vis spectrophotometer (or microplate reader) over a wavelength range of 300–800 nm (see Note

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Fig. 3 Transmission spectra of Ag@SiO2. (a) A typical spectrum of the nanocube solution over a wavelength range of 300–800 nm. The red box indicates the location of the quadrupolar LSPR peak. (b) Zoomed-in view of the quadrupolar LSPR peak. The black squares show the raw spectral data. The red line represents the curve fitting with the fifth order polynomial function

14). The spectrum of the blank solution is used for background subtraction. 3. The typical spectrum is shown in Fig. 3a. Depending on the refractive index of the solution, the peak location of the quadrupolar LSPR lies between 430 and 530 nm. Fit the spectrum data near the quadrupolar LSPR peak (15 nm of the LSPR peak location) using a fifth order polynomial function (Fig. 3b). Locate the wavelength for which this peak has maximum optical density. This wavelength value is called the LSPR peak location. 3.4.2 Calibration of Sensor Sensitivity (Glycerol-Water Test)

Measure the LSPR peak location of nanocubes in several standard solutions with different refractive index (RI) to calibrate the sensitivity (i.e., the change in LSPR peak location vs. the change in RI). 1. Prepare five solutions by mixing glycerol and water: (a) 50 wt% glycerol in DDI water (RI ¼ 1.39809). (b) 25 wt% glycerol in DDI water (RI ¼ 1.36404). (c) 10 wt% glycerol in DDI water (RI ¼ 1.34481). (d) 5 wt% glycerol in DDI water (RI ¼ 1.3388). (e) DDI water (RI ¼ 1.33303). 2. Take five 2 mL snap-cap polypropylene microcentrifuge tubes and label the tubes with the respective glycerol-water solutions. Add 100 μL of the stock Ag@SiO2 nanocubes in each tube.

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3. For each tube, add 1900 μL of the corresponding glycerolwater solution prepared in step 1 and re-disperse particles by vortex mixing. 4. Centrifuge the Ag@SiO2 nanocubes for 5 min at 3260  g. Remove the supernatants and add 2 mL of the corresponding glycerol-water solutions again. Sonicate and vortex the solutions. 5. Centrifuge the Ag@SiO2 nanocubes for 5 min at 3260  g. Remove the supernatant. Add only 250 μL of the corresponding glycerol-water solutions this time. Sonicate and vortex the solution. 6. Follow the LSPR measurement protocol (see Subheading 3.4.1) to identify the LSPR peak location for each glycerolwater solution. 7. Fit a line to the data (RI vs. LSPR peak locations) using linear regression. The slope of the line represents the sensitivity of the sensor (LSPR peak shift (in nm) per RI unit). 3.4.3 Protein Binding Density Calibration (Streptavidin-Biotinyl Lipid Calibration)

1. Prepare a 3 mg/mL small unilamellar vesicle (SUV) solution containing 89 mol% DOPC, 10 mol% DOPS, and 1 mol% biotinyl-capped PE as reported in Subheadings 3.3.1 and 3.3.2. 2. Follow the protocol reported in Subheading 3.3.3 to coat the nanocubes with the desired lipid bilayer. 3. Pipette 200 μL of the lipid bilayer coated Ag@SiO2 nanocube solution into snap-cap microcentrifuge tubes. Mix streptavidin in 1 PBS solution with the lipid bilayer coated Ag@SiO2 nanocube to reach the desired streptavidin concentrations (0.0 nM, 4.5 nM, 9.0 nM, 18.0 nM, 22.5 nM, 29.3 nM, 36.1 nM, and 45.1 nM). Vortex the tubes well and incubate for 30 min. 4. Follow the LSPR measurement protocol reported in Subheading 3.4.1. Record the LSPR peak location at each streptavidin concentration. The first tube has no streptavidin, so the difference in the LSPR peak locations between each sample and tube 1 is the sample LSPR peak shift. These shifts directly correspond to the mass density of streptavidin that has bound to the sensor. 5. Fit a line to the data (streptavidin concentration vs. LSPR shift) using linear regression. The slope may be used to determine the amount of protein binding (mass density) (see Notes 15) and 16.

3.5 Analyses of Lectin Binding

1. Prepare the lipid bilayer coated Ag@SiO2 nanocubes with the desired compositions of glycolipids or neoglycolipids using the

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protocols reported in Subheading 3.3 (see Note 17). A lipid bilayer without glycans should be prepared as the baseline. 2. Mix the lipid bilayer coated sensors with lectins of interest or biological samples (e.g., serum, culture media, etc.) at the desired concentrations (see Note 18). 3. Measure the transmission spectra of the nanocube solutions and the blank solution (i.e., the solution without nanocube sensor) using a UV-Vis spectrophotometer (see Note 19). The transmission spectra of blank solutions should be measured for background subtraction. Follow the LSPR measurement protocol reported in Subheading 3.4.1 to identify the LSPR peak location. 4. Calculate the difference of the LSPR peak locations between each sample and the baseline (i.e., the lipid bilayer sample without glycans). The LSPR shift is proportional to the density of lectins bound to the lipid bilayer. The slope calculated in Subheading 3.4.3 (streptavidin calibration) can be used to estimate the mass density of bound lectins. 5. Users can repeat steps 3 and 4 for a time course analysis or measure the LSPR shift at a single time point. Due to photobleaching, the conventional fluorescence-based approach is not appropriate for kinetic measurements. LSPR measurement is a label-free detection technique; thus, it can monitor lectin binding kinetics with small time intervals.

4

Notes 1. The quality of PD is critical. To minimize contamination, pour PD into a disposable container (e.g., 50 mL falcon tubes). Do not insert the pipette into the bottle. The opened bottle should be stored in a moisture-free environment, such as a desiccator and used within a week. 2. PD is highly viscous. Disposable serological pipettes are a convenient way of measuring PD volumes. 3. After synthesis, the round-bottom flask should be cleaned with freshly prepared aqua regia. 4. The quality of nanocube sensors is highly sensitive to the reaction temperature. A hot plate with precise temperature control is required. 5. The typical average size of nanocube sensor is ~95–115 nm. Size variation among different synthesis batches could lead to variable sensitivity; thus, sensor calibration (glycerol-water test, Subheading 3.4.2) is required for each batch. The typical

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sensitivity of bare nanocube (without silica shell) is 230  36 nm LSPR shift per RI unit. 6. Poor temperature control and contamination of chemicals could lead to increased polydispersity. The size distributions can be directly observed by scanning electron microscope (SEM), but users can also confirm the sensor quality by observing transmission spectra. The LSPR peak (Fig. 3b) is broader when the particles are polydisperse. This parameter can be quantified by calculating the line width of the LSPR spectrum. For a good nanocube sensor batch, the full width at halfmaximum of the quadrupolar LSPR peak should be around 70 nm. 7. During the washing steps, flexible angle centrifuge rotors can reduce the attachment of particles to the side walls of falcon tubes, minimizing particle loss. 8. LSPR sensors only detect the change of refractive index near the metal surface. A thick layer of silica could reduce the sensitivity. However, if the silica layer is too thin, silica may not uniformly cover all the silver surfaces. The incomplete coverage could result in poor coating of lipid bilayers. We prefer to control the thickness of silica layer at 3–5 nm. The thickness and uniformity of the silica shell can be observed by transmission electron microscopy (TEM). Users can also confirm sensor quality by sensitivity measurement (glycerol-water test, Subheading 3.4.2). The typical sensitivity of Ag@SiO2 is 197  27 nm LSPR shift per RI unit. 9. To achieve the desired lipid compositions, we have to calculate the required volume of each stock lipid solution. Groves and his coworkers have established a Microsoft Excel spreadsheet to assist in the calculation. This spreadsheet can be found in the supplementary information of their publication [25]. 10. The lipids should be dissolved in the organic solvents suggested by the vendors. Common solvents are chloroform and chloroform/methanol/water mixture. Piston pipettes should be used to measure the volumes of volatile solvents. 11. In Subheading 3.3.3, the total volume of lipid bilayer coated Ag@SiO2 solution is sufficient to conduct over 100 independent measurements in a 384-microwell plate (10 μL sample per well). All the volumes can be proportionally scaled up or down to meet the needs of the experiment. 12. Supported lipid bilayers form spontaneously upon mixing Ag@SiO2 in a SUV solution. This vesicle fusion process needs to be conducted in buffers. Users can use common biological buffers, such as phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and HEPES-buffered saline. However, the buffer should not contain proteins. Additional proteins (e.g., BSA for

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blocking non-specific adsorption) would physically adsorb to the nanocube sensor surface and reduce the coverage of lipid bilayer. Proteins should be added only after lipid bilayer coating. 13. After forming supported lipid bilayers on nanocube sensors, lipid molecules can stabilize sensor suspension via steric repulsion. Without a sufficient coverage of lipid bilayer, nanocube sensors would aggregate quickly in buffers. Thus, the nanocube solution would become transparent in 1 h. Users can quantify the aggregation by observing the reduction of spectral optical density (OD). This is a simple indicator to confirm whether the lipid bilayer has been coated properly. 14. The OD depends on the particle concentration and the optical path length in the UV-Vis spectrometer, but it does not affect the location of the LSPR peak. However, a lower OD could lead to a lower signal-to-noise ratio and hence reduce the precision of LSPR peak location [23]. To achieve sufficient signal without wasting nanocube sensors, the optimal OD of the LSPR peak should be between 0.1 and 0.3. The OD can be adjusted by changing particle concentrations (dilute or concentrate stock particle solutions). OD can also be adjusted by changing the optical path length in the spectrometer. For the standard UV-vis spectrometer, the optical path can be changed by using different optical cuvettes. If a microplate reader is used to measure the transmission spectra, adding more solution or using a microplate with narrower wells can increase the length of the optical path. 15. In streptavidin-biotinyl lipid calibration, we titrate streptavidin concentrations to vary the protein coverage on nanocube surfaces. We assume that the streptavidin-biotin binding process is fast and irreversible, and that no unbound streptavidin remains in any of the samples. Our prior publication used four independent methods, including streptavidin titration, biotin titration, fluorescence assay, and fluorescence correlation spectroscopy (FCS), to confirm that this hypothesis is valid for estimating bound protein density [23]. 16. The average streptavidin surface density on nanocubes is evaluated by using the DOPC lipid footprint in lipid bilayers (0.72 nm2) [26]. In the protocol (Subheading 3.4.3), 20 μL of 3.79 μmol/mL total lipids is added to the nanocube solution, resulting in a 2 mL solution of lipid bilayer coated nanocubes with 82.2 cm2 surface area of lipid bilayer per mL.

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 2 cm Total surface area ¼ 82:2 mL      lipid 20 μmol  NA ¼ 3:79  2000 mL mol     nm2 1 surface lipid   0:72 2 surface lipid lipid Since it is a bilayer, the number of lipid molecules should be divided by a factor 2. If Cs (mg/mL) of streptavidin is in the tube, we estimate the bound protein mass density by: mg
 mg  C s mL ¼ Bound protein density 2
cm2 82:2 cm mL The bound protein density and LSPR shift should have
a ng linear relationship when bound densities are below 30 cm 2 . The slope of this line is the calibration value for future lectin binding experiments using the same batch of sensors. At high protein surface density, the jamming effects become significant (i.e., a surface bound protein excluding the next protein binding at neighboring ligands); thus, the linear relationship is no longer applied. 17. Although users can mix glycolipids, neoglycolipids, phospholipids, sphingolipids, and cholesterols at any composition, the resultant lipid bilayer may not be stable. A significant number of charged molecules (e.g., sialic acids) can lead to an unstable lipid bilayer. We typically use neutral phosphocholine (PC) lipids as the major components in lipid bilayers (e.g., 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), or L-α-phosphatidylcholine (egg-PC)). When we increase the molar ratios of other molecules, we reduce the amount of these neutral lipids. Typically, lipid bilayers remain stable when up to 20 mol% of neutral glycan molecules present. However, lipid bilayers may become unstable if there are over 10 mol% of charged glycans. If users require a higher molar ratio of charged glycans in lipid bilayers, oppositely charged phospholipid molecules should be included to maintain stability. Common positively charged lipids are 1-palmitoyl-2oleoyl-sn-glycero-3-ethylphosphocholine (EPC) and 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). Common negatively charged lipids are 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and 1,2-dioleoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (DOPG). As described in Sect. 4 (see Note 13), a rapid reduction of OD would be observed if the desired lipid bilayer is unstable. Users can use the OD change as an indicator to confirm the stability of the lipid bilayer.

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18. Biological samples should not contain reducing agents, such as dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP), that are often used to stabilize cysteine residues. The silica shells of Ag@SiO2 particles are porous. These small molecules can diffuse through the shell, react with the silver core, and influence the LSPR measurement. 19. Transmission spectra can be measured by any kind of commercial UV-Vis spectrometer. Monochromator-based UV-Vis spectrometers require a significant amount of time to scan a single spectrum over a wavelength range of 300–800 nm. To reduce the measurement time, users can scan a narrow wavelength range (25 nm of the quadrupolar LSPR peak). The best way to resolve the time issue is using a CCD-based spectrometer (e.g., BMG LABTECH or Ocean optics), which can capture a full wavelength range spectrum within a second. Such high-speed spectral capture will allow users to monitor a large glycan array or monitor real-time glycan binding kinetics.

Acknowledgments The authors thank the support from National Science Foundation (award number: CHE-1904784). References 1. Varki A (2017) Biological roles of glycans. Glycobiology 27(1):3–49 2. Krishnan P et al (2017) Hetero-multivalent binding of cholera toxin subunit B with glycolipid mixtures. Colloids Surf B Biointerfaces 160:281–288 3. Choi HK et al (2019) The influence of heteromultivalency on lectin-glycan binding behavior. Glycobiology 29(5):397–408 4. Lee D et al (2018) Kinetic Monte Carlo modeling of multivalent binding of CTB proteins with GM1 receptors. Comput Chem Eng 118: 283–295 5. Lee D et al (2018) An integrated numerical and experimental framework for modeling of CTB and GD1b ganglioside binding kinetics. AICHE J 64(11):3882–3893 6. Worstell NC et al (2018) Hetero-Multivalency of Pseudomonas aeruginosa lectin LecA binding to model membranes. Sci Rep 8(1):8419 7. Cervin J et al (2018) GM1 gangliosideindependent intoxication by cholera toxin. PLoS Pathog 14(2):e1006862

8. Wands AM et al (2015) Fucosylation and protein glycosylation create functional receptors for cholera toxin. eLife 4:e09545 9. Wands AM et al (2018) Fucosylated molecules competitively interfere with cholera toxin binding to host cells. ACS Infect Dis 4(5):758–770 10. Kirkeby S (2014) Cholera toxin B subunitbinding and ganglioside GM1 immunoexpression are not necessarily correlated in human salivary glands. Acta Odontol Scand 72(8):694–700 11. Vasile F et al (2014) Comprehensive analysis of blood group antigen binding to classical and El tor cholera toxin B-pentamers by NMR. Glycobiology 24(8):766–778 12. Acheson DW et al (1996) Translocation of Shiga toxin across polarized intestinal cells in tissue culture. Infect Immun 64(8): 3294–3300 13. Karve SS, Weiss AA (2014) Glycolipid binding preferences of Shiga toxin variants. PLoS One 9(7):e101173 14. Hurley BP, Thorpe CM, Acheson DWK (2001) Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil

Nanocube-Based Fluidic Glycan Array transmigration. Infect Immun 69(10): 6148–6155 15. Malyukova I et al (2009) Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis. Am J Physiol Gastrointest Liver Physiol 296(1): G78–G92 16. Schu¨ller S, Frankel G, Phillips AD (2004) Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture. Cell Microbiol 6(3):289–301 17. Imberty A, Varrot A (2008) Microbial recognition of human cell surface glycoconjugates. Curr Opin Struct Biol 18(5):567–576 18. Holmner Å, Mackenzie A, Krengel U (2010) Molecular basis of cholera blood-group dependence and implications for a world characterized by climate change. FEBS Lett 584(12): 2548–2555 19. Ramani S et al (2016) Diversity in Rotavirus–Host Glycan Interactions: A “Sweet” Spectrum. Cell Mol Gastroenterol Hepatol 2(3): 263–273 20. Rodighiero C et al (2001) A cholera toxin B-subunit variant that binds ganglioside GM1

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but fails to induce toxicity. J Biol Chem 276(40):36939–36945 21. Worstell NC, Singla A, Wu HJ (2019) Evaluation of hetero-multivalent lectin binding using a turbidity-based emulsion agglutination assay. Colloids Surf B Biointerfaces 175:84–90 22. Sethi A et al (2019) Cell type and receptor identity regulate cholera toxin subunit B (CTB) internalization. Interface focus 9(2): 20180076 23. Wu HJ et al (2012) Membrane-protein binding measured with solution-phase plasmonic nanocube sensors. Nat Methods 9(12): 1189–1U81 24. Worstell NC et al (2016) Binding cooperativity matters: a GM1-like ganglioside-cholera toxin B subunit binding study using a Nanocubebased lipid bilayer Array. PLoS One 11(4): e0153265 25. Nair PM et al (2011) Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. Nat Protoc 6:523 26. Lindblom G, Or€add G (2009) Lipid lateral diffusion and membrane heterogeneity. Biochim Biophys Acta 1788(1):234–244

Part II Glycan Microarray Data Extraction, Processing and use for Measurements

Chapter 5 General Strategies for Glycan Microarray Data Processing and Analysis J. Sebastian Temme and Jeffrey C. Gildersleeve Abstract Glycan microarrays provide a high-throughput technology for rapidly profiling interactions between carbohydrates and glycan-binding proteins (GBPs). Use of glycan microarrays involves several general steps, including construction of the microarray, carrying out the assay, detection of binding events, and analysis of the results. While multiple platforms have been developed to construct microarrays, most utilize fluorescence for detection of binding events. This chapter describes methods to acquire and process microarray images, including generating GAL files, imaging of the slide, aligning the grid, detecting problematic spots, and evaluating the quality of the data. The chapter focuses on processing our neoglycoprotein microarrays, but many of the lessons we have learned are applicable to other array formats. Key words Glycobiology, Glycan Microarray, Data Analysis, Glycan-Binding Protein, High-throughput screening, Carbohydrates, Lectins

1

Introduction Glycan-binding proteins (GBPs) are capable of selectively recognizing carbohydrates and play significant roles in many biological processes, including cell-cell interaction, pathogen-host invasion, host immune recognition and response, cell signaling, and cancer [1–6]. Glycan-binding proteins are often divided into two groups: antibodies (immunoglobulins) and lectins (all carbohydratebinding proteins other than antibodies). Lectins are the largest class of glycan-binding proteins and are found in all phylogenetic kingdoms. Examples of lectins functioning in biological homeostasis and immune regulation include the transmembrane calciumdependent (C-type) lectin asialoglycoprotein receptor (ASGPR), which clears asialoglycoproteins from circulation [7], and the secreted galectin-3 which binds to the N-linked glycans on T cell receptors and functions to regulate immune activation [8, 9]. Additionally, the immune system can generate a repertoire of glycanbinding immunoglobulins in response to abnormal glycosylation

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_5, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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motifs and densities found on infectious diseases and cancerous tissues. Several broadly neutralizing antibodies (bnAbs) to HIV, such as 2G12, bind to the dense mannose coating on the surface of the HIV envelope spike glycoprotein gp120 [10–12]. Glycanbinding immunoglobulin Gs (IgGs) are in various stages of clinical development and include the monoclonal antibody (mAb) Unituxin, which has been FDA approved for the treatment of pediatric neuroblastoma [13]. Unituxin selectively binds to malignancies which overexpress the glycolipid GD2 [14]. The critical roles that lectins and immune-derived glycan-binding proteins play in biological systems have garnered widespread interest in GBPs as potential diagnostic or therapeutic agents. However, because most cell surfaces are decorated with a complex mixture of carbohydrates, therapeutic targeting of atypical carbohydrates on cancer cells or pathogens must be selective and not bind to glycans found on non-diseased human cells. Unfortunately, the structural diversity, lack of glycan-specific markers, glycosylation heterogeneity, and post-translational origin of glycans make them a difficult class of antigens to study [15, 16]. Methods to rapidly analyze the specificity of glycan-binding proteins are critical for identifying natural ligands, developing inhibitors/probes, and understanding protein function. The glycan microarray allows for the high-throughput profiling of a lectin or antibody’s selectivity and binding strength against many carbohydrate structures, sizes, and densities. Several glycan microarray platforms have been developed [17–22]. Glycans can be directly conjugated to a slide’s surface or can be printed as multivalent glycoconjugates, such as neoglycoproteins, glycodendrimers, or glycopolymers. The glycans can be sourced from both organisms and chemical or chemoenzymatic synthetic routes. The selected slide substrate and reactive moiety depends on the chosen conjugation strategy between the glycan or glycoprotein and the slide. Microarrays can be printed to accommodate hundreds to thousands of separate components spanning a broad range of chemical structures, allowing for the high-throughput profiling of multiple samples across hundreds of unique structures. Our group has developed a microarray composed primarily of multivalent neoglycoproteins, along with some natural glycoproteins [23, 24]. Neoglycoproteins are produced by conjugating multiple copies of a particular glycan or glycopeptide via a non-natural linkage to a protein carrier, such as bovine serum albumin [25, 26]. Our current glycan microarray has 738 components, including a variety of N-linked and O-linked glycans, glycolipid glycans, glycopeptides, glycosaminoglycans, non-human glycans, and glycoproteins. The challenges and procedures involved in printing microarrays across various platforms have been previously discussed in detail [22]. The purpose of this chapter is to provide a detailed method to

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aid in the processing of microarray images. The protocol below will focus on details related to processing our neoglycoprotein microarrays but will also include general information for processing microarray data. Generating GAL files, imaging of the slide, aligning the grid, detection of problematic spots, and quality control will be discussed.

2

Materials

2.1 Reagents and Materials

1. Array components printed on SuperEpoxy 2 microarray substrate slides (ArrayIt) or 2D-Epoxy functionalized glass slides (PolyAn 104 00 221). Components are printed in a suitable print buffer containing either 0.05 μg/mL Atto 532 (Sigma 06699) or 0.5 μg/mL Alexa Fluor™ carboxylic acid, tris (triethylammonium) salt (ThermoFisher A33084). Our most recent neoglycoprotein microarray contains over 700 glycoconjugates. 2. Super Friendly Air-it (Fisherbrand—compressed 1,1,1,2 Tetrafluoroethane). 3. ProPlate® 8-well chambers (Grace Bio-Labs). 4. ProPlate® slide module seal strips (Grace Bio-Labs). 5. Blocking buffer (phosphate buffered saline (PBS): 10 mM NaHPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4 with 3% w/v bovine serum albumin). 6. Lectins, glycan-binding monoclonal antibodies, or serum. 7. Fluorescently labeled secondary reagents for detecting bound lectins or antibodies on the array.

2.2

Equipment

1. Microarray fluorescence scanner (Innopsys, InnoScan 1100 AL Fluorescence Scanner). 2. Eppendorf centrifuge 5810R Equipped with swing bucket rotor (A-4-62). 3. Foodsaver V3835 with vacuum seal bags. 4.
20  C freezer for storage of printed microarrays.

2.3

Software

1. Mapix 8.1.1 (Innopsys—fluorescence scanner software). 2. GenePix Pro 7.3 (Molecular Devices—data analysis software). 3. Microsoft NotePad. 4. Microsoft Excel.

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Methods Use of a microarray involves printing of the array, conducting the assay, imaging the slide, and processing the data. Details regarding printing and the microarray assay can be found in some recent reviews [20–22, 27]. Some information that is relevant to image processing will be mentioned below. For the purposes of current chapter, our descriptions and detailed methods will be referencing microarrays composed of either 668 or 738 neoglycoproteins, glycoproteins, and controls. To print these arrays, we use a total of 6 pins in a 3  2 configuration. Three pins are used to print one full array of components, and the second set of 3 pins prints a second full array. Each pin prints a portion of the array, which is referred to as a subarray or block. In total, each 1 inch by 3 inch slide contains 8 full sets of array components. These arrays can be physically separated by a well module and gasket, allowing one to carry out 8 independent array experiments on each slide. The configuration is illustrated in Fig. 1. To aid with image processing and quality control analysis, we include a soluble print dye in the print buffer for each of our array components (Atto 532 or Alexa Fluor 647). The presence or absence of the dye in each printed spot location can be rapidly evaluated using a fluorescence microarray scanner (see Figs. 1 and 2 for sample images acquired during the pre-assay fluorescent scan). The dyes we have selected wash off the slide during our initial wash and block step of the assay. The microarray assay itself is fundamentally simple, with two main variations. In one version, a glycan-binding protein is labeled with a fluorophore prior to incubating on the array. In a second

Fig. 1 Printing setup for an 8-well array. 6 pins arranged in a 3  2 configuration can print 2 full arrays simultaneously. Each array is composed of 3 subarrays, each printed from a separate pin. The final printed array slide contains 8 full arrays from 24 subarrays. Sample pre-assay fluorescent scans are shown for an array with 738 components

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version, a glycan-binding protein is incubated on the array in a first step, followed by incubation with a fluorophore-labeled secondary reagent that will detect the glycan-binding protein. Examples include fluorophore-labeled anti-mouse IgG to detect mouse IgG and fluorophore-labeled streptavidin to detect biotinylated proteins. A high-resolution fluorescence scanner captures an image of the slide which can be converted to a numerical value that represents the binding of the primary analyte for each of the printed components. It is important to note that many fluorophores can be damaged through photooxidative or oxidative decay mechanisms. Therefore, any assay steps involving fluorophore-labeled GBPs or secondary reagents are typically carried out in the dark. If one prints fluorophores on the array itself, as we do, then printing and storage are also done in the dark. Oxidative damage to fluorophores is especially problematic after completion of the assay when the slide is dry. Ground level ozone has been shown to be a key oxidant [28, 29]. As a result, slides should be imaged shortly after completing the assay, and slides are often kept in ozone-free environments (e.g., using catalytic ozone destroyers) prior to imaging. Some dyes are more prone to oxidative damage than others, so use of more stable dyes can also help minimize this problem. For example, we prefer rhodamine-type dyes over cyanine-type dyes. Printed array slides are stored at
20  C in vacuum sealed bags. 3.1 Creating a GAL File for a 3-Subarray Microarray Format

The basic information used by the software program to assist in data analysis is described in a GAL file. When one loads a GAL file into microarray software, the program will generate a series of circles that define the boundaries of the spots. While a single grid of circular features can be generated to analyze the 3 subarrays within the full microarray, we find that the pin to pin deviations result in adjacent subarrays being slightly misaligned as shown in Fig. 2, where adjacent subarrays are slightly shifted vertically. We

Fig. 2 Pre-assay image of 668 component array. Image scanned at 5 μm resolution. Red boxes surround missing samples that failed to print. Each subarray (block) is outlined by a white box. The step-like misalignment of adjacent subarrays is demonstrated here

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therefore generate a single grid for each subarray. The method presented below details how we generate our *.gal files used to analyze an array that is divided into 3 different subarrays. 3.1.1 Create 3 Blocks with the Appropriate Number of Rows and Columns in GenePix

1. Select the “New Block” icon, then fill in the information in the block info box. For the 738 array, 3 blocks are made in this step, each having 20 columns with a spacing of 210 μm  25 rows with a spacing of 204 μm (total of 1500 features in 3 blocks). The spot diameter is dependent on many factors (see Note 1). For our print buffers, settings, and pins, the initial spot diameter can be set to 75 μm. Spacing is based on printing settings. Final spot diameter will be determined during spot alignment (see Note 1). 2. Save these blocks by selecting “Save Settings As” and save as a GenePix Array List File (*.gal). 3. Open the GAL file in NotePad, then save it as a text file. 4. Open the text file in Excel. The newly opened sheet will contain 5 columns: Block, Row, Column, Name, ID. Rename this sheet as “3-block list.” 5. Open a second Excel sheet named “Feature list,” make a list of all the array components along with three columns. The order of columns should be Pin #, Array Component Name, and Array ID #. Pin # corresponds to Block ID in the Gal file (see Fig. 1). 6. If commas are included in the name, the entire name should be surrounded in double quotation marks, e.g., commas,are,bad should be “commas,are,bad” as .csv format is used in gal file formation. 7. Copy and duplicate this component list so that the feature list now contains two copies of each component (see Note 2). 8. Perform 2 sequential sorts on the “Feature list” sheet list. First by Print ID # smallest to largest, and then by pin ID # smallest to largest. 9. Copy Name and ID values from the “Feature list” for all pin 1 components and paste into the “3-block list” Excel sheet under Name and ID columns. For this grid, there will be 8 blank features in each block (see Note 3). 10. In “3-block list,” fill in cells for empty features with EMPTY in both the name and ID.Removed extra words. This should help. 11. Repeat steps 9 and 10 for pins 2 and 3, making sure to paste pin 2 in block 2 and pin 3 in block 3. 12. When the 3 blocks are finished, copy Excel sheet “3-block list” and paste into a new Notepad file. Save as a *.gal file, e.g., 3block.gal.

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13. Load array list by selecting Folder>Load Array List>3block. gal in genepix. A 3-block array grid will appear. 14. Move the 3 blocks to the top of the screen. 15. Select the first block, click on the “Replicate Block Mode” tab, and make 7 copies of block 1 in a vertical orientation. 16. Repeat step 15 for blocks 2 and 3. 17. Save the completed array list with 24 blocks via File>Save Settings As. . . > 24block.gal. 3.2 Pre-Assay Scan of the Microarray Slide

The pre-assay scan of the array slide is the initial QC for the microarray and serves as a template for aligning the array grid after the assay is completed. Printed spots can be evaluated using a light microscope to check the integrity of the print, but we find it more convenient to include a soluble print dye. This scan ensures that all components are printed and provides valuable information about the quality of each printed component. It is recommended that the array slide be scanned at the same resolution and line speed as the post-array scans used for analysis. 1. Remove array slide boxes from freezer and pre-warm to RT for 30 min prior to opening. Slides should be kept in the dark during all incubation steps (see Note 4). 2. Turn scanner on and connect scanner to computer 15 min prior to use to allow time to warm up lamps. 3. Open vacuum seal bag and remove the room temperature slide from array box. Visually inspect array slide for dust and particulates. If particulates are visible, remove using a stream of compressed 1,1,1,2-tetrafluoroethane (TFE). 4. Insert contaminant-free slide into the scanner (top side up). 5. Use the slide read-loader tool to discover the slide (see Note 5). 6. Scan slide using maximum PMT setting or gain setting of R100G100. 7. Export the auto-saved image to a flash drive for grid alignment and data analysis. 8. Remove array slide from scanner and mount an 8-well ProPlate slide module.

3.3 Blocking the Glycan Microarray Slide

We have previously published methods and procedures detailing the microarray assay [30–32]. For this chapter, we would like to highlight the importance of properly blocking the microarray slide. Blocking the slide surface reduces background binding and washes away the fluorescent print dye used in the print buffer. However, as shown in Fig. 3, failure to apply the block gently can result in comet-tailed spots.

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Fig. 3 Comet-tailed features result from improper blocking technique. Assay performed with human serum, IgGs visualized with Cy3-labeled goat antihuman IgG and IgMs visualized with Cy5-labeled goat antihuman IgG. (a) Pre-assay image scanned at 5 μm resolution. (b) Blocking buffer applied to the microarray improperly results in comet-tailed spots radiating from center of droplet impact. (c) Replicate well where the blocking buffer was added slowly to prevent comet tails. Post-assay images scanned at 5 μm resolution

1. Place slide with slide module on a slanted surface and slowly apply 400 μL of blocking buffer using a gel-loading pipette tip (see Note 6 and Subheading 3.6.4). 2. Following the blocking procedure, assay samples according to the experimental procedures. For QC purposes, each batch of slides should be assayed against a set of standard lectins and antibodies. Analysis of this data will be covered in Subheading 3.7.3. 3.4 Post-Assay Scan of the Microarray Slide

1. Following washing, remove array slide from slide module. 2. Submerge slide in PBST for 5 min. 3. Remove slide from PBST and place slide into an empty 50 mL conical falcon tube. 4. Place slide and counterbalance into swing bucket-equipped centrifuge. 5. Centrifuge for 5 min at 500 rcf and 22  C. 6. Remove dried slide from the centrifuge and place into the scanner. 7. Use the slide read-loader tool to discover the slide (see Note 5). 8. Scan slide using an initial gain setting of R15G10 (see Note 7). 9. Repeat scan at a lower gain setting (e.g., R3G1) to ensure that all signals are below the instrument’s maximum of 65,535 RFU. 10. Export the auto-saved images to a flash drive for data analysis.

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Fig. 4 Cartoon representation of microarray grid alignment steps. (a) Initial grid alignment after opening the gal file. (b) Grid alignment following coarse block correction. Features that are absent should be marked as missing (circle marked with an X). (c) Final alignment and feature resizing 3.5 Grid Alignment and Feature Sizing

The first step in the analysis of an array image is locating and defining the spots. The location and size of the spots is largely defined by the printing layout, conditions, and choice of pins (if pin printing is used). When quantitating the fluorescence signals for each spot, the program will use pixels inside the spot boundaries and consider pixels outside the spot boundaries to be background. Therefore, defining the spot locations and size are critical for the analysis of microarray data (see Note 8). We use several steps to achieve precise positioning of the spot boundaries on the actual spots. After initial loading of the gal file, the grid will be off from the printed array (see Fig. 4a) and will need to be coarsely relocated to more precisely cover the printed spots from the pre-assay image. The features in the grid then undergo an initial repositioning over the pre-assay image and the presence of all printed spots is confirmed, the resulting alignment is shown in Fig. 4b. The soluble print dyes added to our print buffer are crucial to our ability to align features over the printed spots and to control for missing components or print errors. After the assay is complete and a final image is captured, fine alignment and resizing of individual features within the grid is finalized (Fig. 4c).

3.5.1 Coarse Alignment to the Pre-Assay Image

1. Open the pre-scan image first and then load the initial GAL file. 2. Under options tab, click on “File Open” change default scaling to 5 μm/pixel, or match scanned image resolution. 3. Under options tab, click on “Alignment” deselect resize features during alignment. 4. Open pre-assay scan image Images. . . > R100G100pre.tif).

in

GenePix

(File>Open

5. Load 24 block GAL file by selecting File>Load Array List>24block.gal. 6. Determine the size of a representative feature. SMP2 pins that we print with generally print 65–75 μm spots.

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7. Adjust all spot diameters to 70 μm by opening block properties, adjust feature diameter to 70 μm and select apply to all. 8. Coarsely align the first block to the top left of the first printed grid and hit F5 to reposition and roughly align all features in the block (see Note 9). Repeating the F5 command may assist in further improving the automated feature alignment. 9. Check each feature in block 1 grid for alignment. 10. If a spot is missing, flag the feature by selecting the feature and marking as bad (type A for bad). 11. Save a new settings file (File> Save Settings As. . . > preassay. gps) (see Note 10). This file has the GAL file as well as other settings. 12. Repeat steps 7–10 for the remaining 23 blocks, overwriting the file as you progress. 3.5.2 Coarse Grid Alignment to the PostAssay Image

Due to slight deviations resulting from variations in loading of the slides into the scanner, images before and after an assay are not perfectly overlapped. Therefore, the grids aligned on the pre-scan image must be coarsely adjusted to the final post-assay image. 1. Open the post-assay array image with the highest gain setting (File>Open Images. . . > R15G10.tif). 2. Open the pre-assay scan Settings. . . > preassay.gps).

settings

file

(File>Open

3. Save a new settings file (File> Save Settings As. . . > R15G10. gps). 4. To increase signal to noise and contrast for data analysis, click on! icon (Auto Scale Brightness and Contrast). 5. Align each block to the top left of each subarray (see Note 11). 3.5.3 Fine Alignment and Feature Resizing of the Post-Assay Image

The automatic feature alignment tool employed in Subheading 3.5.1 is often capable of positioning a feature within 0–2 pixels from dead center (in the case of a 5 μm scan, 1 pixel is 5 μm); however, this tool may produce features that could be off by 5 or more pixels. Therefore, the alignment of features is verified manually after the automatic processing and any required adjustments are made prior to data processing. In addition to aligning the spots, it may be necessary to resize the spots (see Note 12). Genepix software has a process to automatically adjust the feature sizes, but we find that it is not very effective and often overestimates the size of the features within the grid. A global feature diameter that matches a pin’s typical print diameter is applied to the settings file and the diameter of individual features is verified and adjusted during the feature repositioning procedure. The following is the general procedure used when adjusting features over good or normal

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Fig. 5 Representative images of typical and abnormal microarray spots. (a) Normal spots in duplicate. (b) Lint or other debris deposited on the array. (c) Scratched microarray surface following assay leads to spot-spot variability. (d) Irregular drying caused by the coffee ring effect. (e) A “donut” on an older array. (f) An example of a “comet tail” cause by over-aggressive pipetting during the blocking step

spots (Fig. 5a). It is important to recognize non-uniform or bad spots (Fig. 5b–f), which will be discussed in detail in Subheading 3.6. 1. Begin feature alignment and resizing by selecting block 1 and typing 1 to zoom into top left corner of the grid. Center each feature over the positive spots and resize by maximizing signal and minimizing background. In Genepix, features can be moved using arrows and resized using Ctrl + " to increase diameter and Ctrl + # to decrease diameter. In general, the circular feature should be completely within the visualized spot, having no background in the measurement (see Fig. 4c). It is also important to make sure that a large majority of the spot is within the feature and that a feature is not too small (see Note 13). The average fluorescent intensity of a properly sized feature should not change with slight adjustment in size. 2. Move to the top right of grid 1 by typing 2, repeating the adjustments for any positive hits. Move to the bottom left and right of grid 1 by typing 3 and 4, respectively, repeating the centering and sizing for any positive hits. Depending on the

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number of positive components, precise alignment of each block can take 1–30 min. 3. Overwrite the settings file (File> As. . . > R15G10.gps) (see Note 10).

Save

Settings

4. Repeat steps 1–3 until all blocks are finished. 3.6 Identification of Problematic/Artifact Spots

One of the most critical and difficult steps in microarray data processing is identifying and handling of non-uniform microarray spots. In this section, we will highlight several types of problematic microarray artifacts and how to deal with them.

3.6.1 Scratches, Lint, and Other Artifacts that Obscure Spots

It is relatively common for the array surface to have pieces of lint, hair, or dust that give rise to high levels of fluorescence (Fig. 5b). If these objects obscure a spot and cannot be removed with compressed gas, that spot should be flagged as “bad” using the software and removed from analyses. Scratches can also occur on the array surface, especially if a pipette tip contacts the surface during the assay. Scratches can remove fluorescence signals from part or all of a spot (Fig. 5c). Again, these spots should be flagged as “bad” and removed from further analyses.

3.6.2 Large Differences for Replicate Spots

Some variation in signal intensity from spot-to-spot is to be expected, but occasionally, the post-assay fluorescence intensities of two seemingly “identical” spots are vastly different. This situation is complicated, since one spot may have a very strong signal while another spot has essentially no signal. The most common cause of non-equivalent spots results from the printing of the array itself, wherein a replicate spot is either not printed or mis-printed. There are several strategies to address this type of problem. First, assess the quality of the print batch. Check the pre-assay image of the array and ensure that replicate spots were printed and are not contaminated by dust or other particulates. If the pre-assay scan image clearly shows a missing or damaged spot, mark the problematic feature as bad or missing and continue with the array analysis using the other spots in the analysis. In addition to QC, another approach is to print 3–6 replicate spots and then determine the outlier. Alternatively, one can analyze the sample of interest on two or more separate microarrays to determine the consensus result. Large differences in signal can also be attributed to the effect seen in Subheading 3.6.3. When this occurs, we typically flag both spots and remove them from further processing. If these data are essential, we will repeat the experiment.

3.6.3 Donuts or Ring-like Spots

Ring-like spots, with higher signals around the perimeter and lower signals in the center, are a common problem with microarray data. Modest effects are often seen as a result of the “coffee ring effect” [33, 34]. When the evaporating liquid at the perimeter of the

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deposited spot (at the air-water interface) is replaced by liquid from the center, a buildup of the printed component occurs at the perimeter of the spot. As a result, there can be a higher concentration of array component at the perimeter, leading to signals at the perimeter that are 2–3 times higher than the center. For examples of this type of effect and the resulting spots, see Fig. 5d. Optimization of printing conditions, e.g., printing under a humidified atmosphere, printing with a high enough component concentration to saturate the reactive slide substrate, and the addition of glycerol and detergents to the print buffer, alleviate much of this artifact but it can still occur [35, 36]. We typically process these types of spots in the same way as normal spot, examples of a normal spot are shown in Fig. 5a. In some cases, the ring can be much sharper and involve a much larger difference in intensity between the outer perimeter and the center. This type of donut artifact is characterized by a sharp saturated ring at the perimeter of the spot with a typical homogenous signal or no signal at the center of the printed spot. For example, pixels at the perimeter might be 50,000 RFU while pixels in the center are only 500 RFU. Because of the extreme difference, we believe this situation is distinct from coffee ring effects. Our current explanation is that these types of spots arise as a result of damage to the protein at the air-water interface during the printing and long-term storage of a microarray. For a droplet on a slide, most of the air-water interface is well above the surface of the slide; however, at the perimeter, the air-water interface contacts the slide surface. When the slide is blocked, the damaged protein at the perimeter sticks to the surface and is retained while the damaged protein in the middle is washed away (see Note 14). These oxidized/damaged proteins can have altered properties and can be much stickier than intact proteins, giving rise to high signals at the perimeter that are not related to glycan recognition. When these sharp-edged spots are observed, the perimeter of the spot is excluded from analysis by focusing the feature diameter on the central core of the spot. For examples of this type of damage and the resulting spots, see Fig. 5e. Although the exact nature of the damage has not been determined, we believe it involves oxidation (e.g., by ozone) and/or denaturation of the protein during the printing and storage of the slides. In practice, the problem can largely be eliminated by filtering the air that goes into the humidifier (and then into the microarray instrument) through a carbon filter to remove ozone and other volatile organic materials. Passing the air through a carbon filter also prevents loss of signal for the printed dye-BSA conjugates and the soluble dye in our print buffer. As additional support for ozone as a key mediator of damage, printing of BSA that has been treated with ozone results in high signals, whereas untreated BSA has low

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or no signal. Storage of slides at low temperature and in vacuum seal bags is also important to minimize additional oxidation/damage after the printing step. 3.6.4 Comet Tails

A common problem with microarray data is the formation of comet tails, see Fig. 5f. We observe this type of artifact when the blocking solution is applied to the microarray slide with too much force, causing excess material in a printed spot to contact the surrounding epoxide-coated slide substrate before the blocking protein is able to saturate the non-printed slide surface, resulting in spots that appear as comet tails. Figure 3a shows the pre-assay image of an array slide which was then blocked by applying a droplet of blocking buffer directly to the array surface with too much force. Following the assay, the array was imaged (Fig. 3b), resulting in spots resembling comets radiating from the central impact “crater” caused by the blocking droplet. For comparison, Fig. 3c shows a replicate well where the blocking buffer was added slowly in the corner of the well to prevent comet tails. When analyzing spots with comet tails, the feature should be centered on the body of the “comet” and the “tail” should always be ignored. To avoid these irregularly shaped spots, pipette blocking solution slowly into the corner of the well using a gel-loading tip as described in Subheading 3.3 step 1. In some situations, we observe a similar type of artifact that is derived from a pin printing error. For example, the pin may not have been moved up far enough from the array surface before being moved laterally to the next location, essentially dragging/streaking liquid towards the next spot location. If the comet tail occurred during the printing step, the tail will be present in the pre-scan image. To avoid this type of irregularity, ensure that the pins are not too close to the surface when moving laterally.

3.7 Processing and Analysis of Microarray Data

Once the grids have been aligned and the features have been resized, the next step is to convert the image into raw fluorescence data and export it into data processing software, such as Excel.

3.7.1 Extracting Data from GenePix into Excel

1. Select the data button in GenePix to convert features into numbers. 2. For a single green fluorescent measurement. Under the Results>Data Types tabs, Select data types Name, Flags, ID, Block, Column, Row, 532 Median-532 Background, 532 Median, and 532 Background. Add additional laser scans as dictated by the experiment and chosen secondaries (see Note 15). 3. Export results as a raw Results. . . > results.txt file.

text

file

by

File>Export

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1. Import results.txt file into an Excel sheet. 2. Assemble a second sheet in Excel containing formulas that remove flagged features values and average each component’s median-background (see Supporting Excel File Sheet for examples and useful formulas). 3. Copy the formula-containing sheet into a new sheet, highlight all formula-containing cells and paste as values. 4. Sort to remove any blank or false values in the medianbackground final column, and sort to remove any empty features/components in the name list. 5. Select all data for each well and copy the data into a sheet containing the array list. This will serve as the final data. 6. Repeat this process for any additional scans at other PMT settings of interest.

3.7.3 Analysis of Data Quality: Batch QC

We take a variety of steps to ensure high-quality data. As mentioned above, we incorporate a soluble dye into our print buffer to aid in assessing print quality. In addition, we print a variety of control spots to aid in analyses. Some examples include the unmodified carrier proteins HSA and BSA as negative controls, immunoglobulins from multiple species as positive controls for the secondary antibodies, and fluorescently labeled BSA controls, which aid in grid alignment, RFU normalization, and PMT optimization. Lastly, we assay one slide from each print batch using a standard set of 3 lectins (ConA, WGA, and HPA) and 2 antibodies (T36 to blood group A and CLCP19-B to blood group B) to evaluate consistency from one print batch to another. These proteins are assayed in multiple wells to assess well-to-well variability as well as batch-to-batch effects. The set of QC proteins was selected to cover a wide range of glycan components on our array. A thorough QC will illuminate printing issues and any errors made during the source plate setup. The following represents a few simple comparisons which should help to determine the quality of the data. These analyses should be performed on the final data. 1. For replicate data from two separate array wells, select two columns in Excel, plot an xy scatter plot with trendline and R2 value. R2 values should be high, >0.95. 2. If there are any obvious outliers from the trendline, these features should be checked. 3. Data from each QC should be compared to previous QC experiments for obvious differences (see Note 16). 4. Only after a print batch has passed QC, should newly printed slides be used in experimentation. QC should be repeated every 6 months to monitor microarray stability.

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3.7.4 Analysis of Data Quality: Experimental QC

While image quality analysis from Subheadings 3.4 and 3.5 are important, it is also important to recognize and deal with the quality of the numerical data in the Excel file. One of the most important measures taken during the array design to assist in data certification is the incorporation of positive controls. Printed positive controls are critical to control for experimental errors, e.g., a printed human IgG should have a large positive signal if a labeled antihuman IgG-specific secondary antibody is used for fluorescent visualization. Positive controls will also control for number extraction errors. For instance, a high positive value is expected for Cy3-conjugated BSA in the Excel spread sheet. If this is not the case, then there could be an error in the processing or in the grid setup. 1. Sort each data field from highest value to lowest value. 2. Verify that all positive and negative controls are as expected in the new microarray design.

3.8 Normalization and Scaling of Data

4

Normalization and scaling of data is helpful when comparing data from one experiment to another, especially over long periods of time. Over time, the fluorophores on secondary reagents can decrease in fluorescence. Also, a new batch of secondary reagent may have a different number of fluorophores per molecule of secondary reagent. In addition, the performance of a scanner can change, such as before and after maintenance. Adjusting for these factors allows one to more easily compare data from different experiments. We use two approaches. For individual lectins and monoclonal antibodies, we use our Cy3-BSA conjugate spots as a reference signal. When assaying serum samples, our approach is to run a standard serum sample in 1 well of the slide. We determine the average RFU value for a standard group of 17 glycans and then determine the adjustment needed to shift that average value to a predefined value. For example, our predefined average value might be 25,000 RFU. If the average for those 17 glycans for a particular well is 20,000 RFU, then we would scale all data on that slide up by 25%.

Notes 1. Printed spot diameter is dependent on a number of variables including pin size, protein concentration, humidity, printing parameters, and printing buffer hydrophobicity. These factors are held constant, as much as possible, throughout the print to minimize variability; however, some variability comes from the reloading of the contact pins during the course of the print. The pins are loaded with solubilized array components by

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dipping into a source plate; however, a single load is insufficient to print an entire batch of slides. The pins are reloaded several times during the printing of each component. Slides which are printed shortly after a load have large spots (100 μm) and slides which are printed towards the end of a load have smaller spots (70 μm). 2. We often print in duplicate. If additional replicates are required, the grid can be adjusted by replicating this list to match the number of replicates. 3. In the current microarray setup for the full array, the components are printed in duplicate. 246 components per block for a total of 492 spots per block. Therefore, at the end of each block, there are 8 empty components. 4. Slides should be kept in the dark at all times to protect printed fluorescent components and labeled secondary antibodies. Aluminum foil coated boxes are employed to prevent photooxidative damage. 5. ArrayIt slides are not reliably recognized by the InnoScan 1100 AL slide sensor. If this becomes a problem, we recommend applying white out or black sharpie to the lower right-hand side of the slide. Only a few millimeters of the slide need to be marked for the scanner to recognize the slide. Care should be taken to wipe away excess white out to avoid introducing dried residue into the scanner. This is not a problem with PolyAn slides. 6. Blocking buffer is applied to the array by positioning the pipette tip in the bottom left-hand corner of array well and then pipetting the liquid into the well very slowly. Applying block too fast or dropping block onto the printed array from a distance will result in comet tails and other unwanted artifacts. 7. Scan settings are dependent on sample and secondary. The InnoScan 1100 AL Fluorescence Scanner has a maximum RFU of ~65,500 for a saturated spot. It is best to have the strongest signals below this saturation for the most concentrated samples. Low resolution array preview scans will save time in identifying the best starting gain for each slide to prevent having saturated signals. 8. Although microarray printers deposit spots with high precision, some variations from a perfect grid are to be expected. The entire print array can be shifted up, down, left, or right relative to the perfect grid. In addition, alignments of specific rows or columns can be larger or smaller than expected. Finally, individual spots can be shifted slightly from the correct position and/or can have different sizes. Typically, the grid of spots defined by the initial Gal file must be adjusted.

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9. F5 will align all spots within the grid. We find this feature to be very good if the grid is first aligned to the top left of the printed subarray. The application’s algorithm does a poor job if the grid is not first pre-aligned with the subarray. If a spot auto-sizing process is performed, the results are often not acceptable (see Subheading 3.5.3). 10. Frequent saving of the settings file is strongly encouraged. 11. While the features within each grid will be aligned reasonably well following pre-assay scan alignment in Subheading 3.2, each time the slide is scanned, the global grid alignment will be very slightly off due to slide insertion/feeding variability. 12. Most sources of size variability can be controlled during the printing process. For the neoglycoprotein microarray platform printed on an epoxide-coated slide, the spot sizes on a single slide are generally uniform when printed using identical pins. Most sources of spot size variability originate from print buffer components (e.g., detergents and glycerol) and protein concentration, both are kept constant for all printed components. Because of the general uniformity of the spots, it is best to apply a global feature diameter as in Subheading 3.5.3 on the pre-assay scan, and then adjust the size of individual features during the post-array image analyses. 13. During feature resizing and spot alignment, it is good practice to exclude spots that are drastically different in size from adjacent spots. As an example, if a pin is printing spots with an average diameter of 70 μm, then a 25 μm spot indicates that there may be a problem and should be excluded from analysis. The most likely culprit for an undersized spot is that the pin is empty or nearly empty, thus depositing a non-uniform spot that could produce a biased experimental result. 14. Donut artifacts are of significant concern and poorly understood. By examining many spots using high-resolution scans (0.1 μm/pixel), the data seem most consistent with formation of blister-like droplets on the array surface. The apparent damage occurs at the air-droplet interface and results in a thin film, or blister, of damaged material that seems to bind to certain samples or secondary reagents. During the blocking step and subsequent washes, these blisters are often washed away to reveal the unharmed slide surface within a damaged ring. In Fig. 3c, a duplicate-printed component is observed with and without a blister-like cap on the spot. 15. While many data types are extractable (>50 measurements), the (median – background) of each given signal is the preferred measurement that best estimates a feature’s signal for the neoglycoprotein microarray. Depending on the scanner model and the complexity of the experiment, more than one

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fluorophore can be imaged. The InnoScan 1100 AL is capable of scanning up to 3 wavelengths allowing for multiplexed experiments (e.g., Cy3-labeled anti-IgG and Cy5-labeled anti-IgM) to be assayed using a single sample and a single array well. 16. Print-to-print variability can result from pipetting errors in plate setup or degradation or replacement of array components. Any time array components are exchanged, or a new component is added to the array, a QC experiment designed to achieve a positive signal for the components should be employed.

Acknowledgments We thank the Consortium for Functional Glycomics (GM62116; The Scripps Research Institute), X. Huang (Michigan State University), T. Tolbert (University of Kansas), Lai-Xi Wang (University of Maryland), J. Barchi (National Cancer Institute), T. Lowary (University of Alberta), Glycan Therapeutics, Glycohub, and Omicron Biochemicals Inc. for generously contributing glycans for the array. This work was supported by the Intramural Research Program of the National Cancer Institute, NIH. References 1. Lopez PH, Schnaar RL (2009) Gangliosides in cell recognition and membrane protein regulation. Curr Opin Struct Biol 19(5):549–557. https://doi.org/10.1016/j.sbi.2009.06.001 2. Schnaar RL (2004) Glycolipid-mediated cellcell recognition in inflammation and nerve regeneration. Arch Biochem Biophys 426(2): 163–172. https://doi.org/10.1016/j.abb. 2004.02.019 3. Schnaar RL (2015) Glycans and glycanbinding proteins in immune regulation: a concise introduction to glycobiology for the allergist. J Allergy Clin Immunol 135(3):609–615. https://doi.org/10.1016/j.jaci.2014.10.057 4. Raman R, Tharakaraman K, Sasisekharan V et al (2016) Glycan-protein interactions in viral pathogenesis. Curr Opin Struct Biol 40: 153–162. https://doi.org/10.1016/j.sbi. 2016.10.003 5. Poole J, Day CJ, von Itzstein M et al (2018) Glycointeractions in bacterial pathogenesis. Nat Rev Microbiol 16(7):440–452. https:// doi.org/10.1038/s41579-018-0007-2 6. Zanetta JP, Kuchler S, Lehmann S et al (1992) Glycoproteins and lectins in cell adhesion and

cell recognition processes. Histochem J 24(11):791–804. https://doi.org/10.1007/ bf01046351 7. Ashwell G, Harford J (1982) Carbohydratespecific receptors of the liver. Annu Rev Biochem 51(1):531–554. https://doi.org/10. 1146/annurev.bi.51.070182.002531 8. Nangia-Makker P, Hogan V, Raz A (2018) Galectin-3 and cancer stemness. Glycobiology 28(4):172–181. https://doi.org/10.1093/ glycob/cwy001 9. Breuilh L, Vanhoutte F, Fontaine J et al (2007) Galectin-3 modulates immune and inflammatory responses during helminthic infection: impact of galectin-3 deficiency on the functions of dendritic cells. Infect Immun 75(11): 5148–5157. https://doi.org/10.1128/IAI. 02006-06 10. Calarese DA, Scanlan CN, Zwick MB et al (2003) Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300(5628): 2065–2071. https://doi.org/10.1126/sci ence.1083182

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11. Doores KJ, Fulton Z, Huber M et al (2010) Antibody 2G12 recognizes di-mannose equivalently in domain- and nondomain-exchanged forms but only binds the HIV-1 glycan shield if domain exchanged. J Virol 84(20): 10690–10699. https://doi.org/10.1128/ JVI.01110-10 12. Pejchal R, Doores KJ, Walker LM et al (2011) A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science 334(6059):1097–1103. https://doi.org/10.1126/science.1213256 13. Mueller I, Ehlert K, Endres S et al (2018) Tolerability, response and outcome of highrisk neuroblastoma patients treated with longterm infusion of anti-GD2 antibody ch14.18/ CHO. MAbs 10(1):55–61. https://doi.org/ 10.1080/19420862.2017.1402997 14. Yu AL, Gilman AL, Ozkaynak MF et al (2010) Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med 363(14):1324–1334. https://doi. org/10.1056/NEJMoa0911123 15. Cummings RD, Pierce JM (2014) The challenge and promise of glycomics. Chem Biol 21(1):1–15. https://doi.org/10.1016/j. chembiol.2013.12.010 16. Cummings RD (2009) The repertoire of glycan determinants in the human glycome. Mol BioSyst 5(10):1087–1104. https://doi.org/ 10.1039/b907931a 17. Liu Y, Palma Angelina S, Feizi T (2009) Carbohydrate microarrays: key developments in glycobiology. Biol Chem 390:647–656. https://doi.org/10.1515/BC.2009.071 18. Geissner A, Seeberger PH (2016) Glycan arrays: from basic biochemical research to bioanalytical and biomedical applications. Annu Rev Anal Chem (Palo Alto, Calif) 9(1): 223–247. https://doi.org/10.1146/annurevanchem-071015-041641 19. Park S, Gildersleeve JC, Blixt O et al (2013) Carbohydrate microarrays. Chem Soc Rev 42(10):4310–4326. https://doi.org/10. 1039/c2cs35401b 20. Mende M, Bordoni V, Tsouka A et al (2019) Multivalent glycan arrays. Faraday Discuss 219: 9–32. https://doi.org/10.1039/c9fd00080a 21. McQuillan AM, Byrd-Leotis L, HeimburgMolinaro J et al (2019) Natural and synthetic sialylated glycan microarrays and their applications. Front Mol Biosci 6(88):88. https://doi. org/10.3389/fmolb.2019.00088 22. Hyun JY, Pai J, Shin I (2017) The glycan microarray story from construction to applications. Acc Chem Res 50(4):1069–1078.

https://doi.org/10.1021/acs.accounts. 7b00043 23. Zhang Y, Gildersleeve JC (2012) General procedure for the synthesis of neoglycoproteins and immobilization on epoxide-modified glass slides. In: Chevolot Y (ed) Carbohydrate microarrays: methods and protocols. Humana Press, Totowa, NJ, pp 155–165. https://doi. org/10.1007/978-1-61779-373-8_11 24. Campbell CT, Zhang Y, Gildersleeve JC (2010) Construction and use of glycan microarrays. Curr Protoc Chem Biol 2(1):37–53. https://doi.org/10.1002/9780470559277. ch090228 25. Roy R (1996) Syntheses and some applications of chemically defined multivalent glycoconjugates. Curr Opin Struct Biol 6(5):692–702. https://doi.org/10.1016/S0959-440x(96) 80037-6 26. Stowell CP, Lee YC (1980) Neoglycoproteins the preparation and application of synthetic glycoproteins. In: Tipson RS, Horton D (eds) Advances in carbohydrate chemistry and biochemistry, vol 37. Academic Press, New York, pp 225–281. https://doi.org/10.1016/ s0065-2318(08)60022-0 27. Rillahan CD, Paulson JC (2011) Glycan microarrays for decoding the glycome. Annu Rev Biochem 80(1):797–823. https://doi.org/ 10.1146/annurev-biochem-061809-152236 28. Branham WS, Melvin CD, Han T et al (2007) Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements. BMC Biotechnol 7(1):8. https://doi.org/10. 1186/1472-6750-7-8 29. Fare TL, Coffey EM, Dai H et al (2003) Effects of atmospheric ozone on microarray data quality. Anal Chem 75(17):4672–4675. https:// doi.org/10.1021/ac034241b 30. Xia L, Gildersleeve JC (2015) The glycan array platform as a tool to identify carbohydrate antigens. In: Lepenies B (ed) Carbohydratebased vaccines: methods and protocols. Springer, New York, NY, pp 27–40. https:// doi.org/10.1007/978-1-4939-2874-3_3 31. Durbin SV, Wright WS, Gildersleeve JC (2018) Development of a multiplex glycan microarray assay and comparative analysis of human serum anti-glycan IgA, IgG, and IgM repertoires. ACS Omega 3(12):16882–16891. https:// doi.org/10.1021/acsomega.8b02238 32. Zhang Y, Campbell C, Li Q et al (2010) Multidimensional glycan arrays for enhanced antibody profiling. Mol BioSyst 6(9):1583–1591. https://doi.org/10.1039/c002259d

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Chapter 6 Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism Marie Le Berre, Jared Q. Gerlach, Iwona Dziembała, and Michelle Kilcoyne Abstract Half maximal inhibitory concentration (IC50) is a measurement often used to compare the efficiency of various carbohydrates and their derivatives for inhibition of lectin binding to particular ligands. IC50 values can be calculated using experimental data from various platforms including enzyme-linked immunosorbent assay- (ELISA-)type microtiter plate assays, isothermal titration calorimetry (ITC), or glycan microarrays. In this chapter, we describe methods to fluorescently label a lectin, to carry out a lectin binding inhibition experiment on glycan microarrays, and to calculate the IC50 value of a binding inhibitory molecule using GraphPad Prism software. In the example used to illustrate the method in this chapter, IC50 calculation is demonstrated for inhibition of Maackia amurensis agglutinin (MAA) binding to 30 sialyl-N-acetyllactosamine (3SLN) using free lactose. Key words IC50 concentration, IC50 value, Half maximal inhibitor concentration, Glycan microarrays, Inhibition, Haptenic sugar, Lectin, MAA, MAL, Lactose

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Introduction Lectins and other carbohydrate-binding proteins are frequently used for deducing the presence or absence of certain carbohydrate residues or structures on cells, tissues, (glyco)proteins, and other molecules [1–3]. As lectins often have multiple non-carbohydrate binding sites in addition to their carbohydrate binding sites, inhibition of lectin binding by co-incubation with the appropriate free haptenic sugar is used to confirm carbohydrate-mediated lectin binding [4]. Beyond confirmation of carbohydrate-mediated binding, inhibition of lectin binding also provides the possibility of measurement, enabling comparison of lectin and hapten performance. Data from glycan microarrays have been used to quantify various binding parameters including dissociation constant (KD), inhibitory constant (Ki), and half maximal inhibitory concentration (IC50) [1, 5, 6]. IC50 values are often used as convenient

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_6, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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Fig. 1 (a) Inhibition of binding of fluorescently labeled lectin (MAL) to a neoglycoprotein (30 sialyl-N-acetyllactosamine-BSA, 3SLN-BSA) presented on a microarray surface in the presence of free haptenic sugar (lactose). (b) Inhibition of binding of fluorescently labeled 3SLN-BSA to MAL presented on microarray surface in the presence of free lactose. The BSA image is structure 3C03 from the RCSB Protein Data Bank (PDB). The MAL depicted is MAL complexed with sialyllactose (structure 1DBN) from the PDB [7]. Carbohydrates were depicted using Consortium for Functional Glycomics residue symbols in GlycoWorkbench, where the yellow circle is galactose, blue circle is glucose, blue square is N-acetylglucosamine, and purple diamond is Nacetylneuraminic acid (sialic acid) [8]

comparators for assessing the efficiency of carbohydrate-based inhibitors for various potential applications including preventing infection or modulating immune response [1, 6]. In this chapter, we describe a method for calculating IC50 values using data from the inhibition of a lectin binding to a carbohydrate ligand on a glycan microarray platform using the free haptenic sugar (Fig. 1a). The same method can be used to calculate IC50 of the reverse situation, the inhibition of glycoprotein or neoglycoprotein binding to a lectin conjugated to a lectin microarray platform using the free haptenic sugar (Fig. 1b). Maackia amurensis agglutinin (MAA) is widely used to demonstrate the presence or absence of α-(2,3)-linked sialic acid on cells and in tissue and protein preparations. MAA is purified from seeds

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from the legume tree Maackia amurensis and contains two lectins, Maackia amurensis leukoagglutinin (MAL, also known as MAA-I) and Maackia amurensis hemagglutinin (MAH, also known as MAA-II). MAL preferentially binds to 30 sialyl-N-acetyllactosamine (3SLN; Neu5Ac-α-(2,3)-Gal-β-(1,4)-GlcNAc) and SO42-3Gal-β-(1,4)-GlcNAc, while MAH additionally binds to the disialylated tetrasaccharide Neu5Ac-α-(2,3)-Gal-β-(1,3)[Neu5Ac-α-(2,6)-]GlcNAc-, the polysialylated structure Neu5Ac-α-(2,3)-Neu5Ac-α-(2,8)-Neu5Ac-α-(2,3)-Gal-β-(1,4)Glc- and SO42-3-Gal-containing structures [7, 9]. The binding of both lectins to carbohydrates can be inhibited by high concentrations of free lactose (Gal-β-(1,4)-Glc) [9]. In the example used to illustrate the IC50 calculation method in this chapter, various concentrations of lactose were used to inhibit MAA binding to a 3SLN neoglycoprotein printed on a glycan microarray.

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Materials Prepare all solutions in ultrapure water (15.5 MΩ at 25  C) and using analytical grade reagents unless indicated otherwise. Prepare and store all reagents at room temperature unless indicated otherwise. All pipette tips and tubes used should be autoclaved before use or purchased sterilized. Follow all local waste disposal regulations when disposing of waste materials.

2.1 Fluorescently Labeling MAA Lectin

1. Pure unlabeled MAA lectin (or whichever lectin is of interest for the study) (see Notes 1 and 2). 2. 500 mM sodium borate, pH 8.2. 3. High purity lactose (99%, anhydrous or monohydrate) (or appropriate haptenic sugar for selected lectin). Make 20 mM lactose in 500 mM sodium borate, pH 8.2, by weighing 72 mg high purity lactose monohydrate (or 68.46 mg high purity anhydrous lactose) in a sterile 15 mL tube and adding 10 mL 500 mM sodium borate, pH 8.2. Cap the tube and mix by inversion until the lactose is completely dissolved. Sterilize by filtration through a 0.22 μm membrane. 4. Alexa Fluor® 555 succinimidyl ester, 1 mg (ThermoFisher Scientific, Waltham, MA, USA). Before carrying out the labeling reaction, remove the dye vial from the freezer and allow to equilibrate to room temperature for approximately 30 min. Keep the dye vial in the dark at all times and protect from moisture (see Note 3). 5. Anhydrous dimethyl sulfoxide (DMSO) (99.5%) (see Note 4).

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6. Amicon® Ultra-0.5 Ultracel-3 membrane centrifugal filters, 3 kDa molecular weight cut-off (MWCO) (Merck Millipore Ltd., Cork, Ireland). 7. A low salt version of Tris-buffered saline supplemented with Ca2+ and Mg2+ ions (TBS) is used for glycomics microarray experiments (see Notes 5 and 6). TBS: 20 mM Tris–HCl, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH to 7.2 with concentrated HCl. Usually 1 L of an autoclaved 10 TBS stock is made and 100 mL of this 10 TBS stock is used to make 1 L of 1 TBS just before use by diluting with 900 mL of ultrapure water. 8. Sterile 15 mL polypropylene tubes. 9. Sterile amber (or dark opaque) microcentrifuge tubes, 2 mL (for reaction) and 0.5 mL (for aliquots). 2.2 Binding Inhibition Incubations of MAA-AF555 on Glycan Microarray Slides

1. TBS. 2. TBS-T: For 1 L of TBS-T, add 900 mL ultrapure water to 100 mL 10 TBS and then add 0.5 mL molecular biology grade Tween® 20 (0.05%) (see Note 7). 3. Alexa Fluor® 555-labeled MAA (MAA-AF555) of known concentration (from Subheading 2.1). Make a 2 μg/mL stock solution in TBS-T to allow a final incubation concentration of 1 μg/mL. Always handle fluorescently labeled lectins in the dark. 4. Neoglycoprotein and glycoprotein (glycan) microarray slides (or other glycan microarray slides). Three slides will be needed for a triplicate experiment and two or more additional microarray slides will be needed for titration experiments. Glycan microarray slides should be comprised of multiple replicate subarrays on the same slide to facilitate inhibition experiments. Our glycan microarray slides have eight replicate subarrays on one microarray slide. 5. Sterile 15 mL polypropylene tubes. 6. High purity lactose (99%, anhydrous or monohydrate). Make a 200 mM lactose stock dilution in TBS-T by weighing 0.6842 g of anhydrous lactose (adapt quantity according to molecular mass (Mr) if lactose monohydrate is used instead) into a 15 mL sterile polypropylene tube and adding 10 mL TBS-T. Cap the tube and mix by inversion until lactose has fully dissolved. 7. Sterile or autoclaved 0.5 mL microcentrifuge tubes. 8. Series of concentration dilutions of lactose co-incubated with a constant concentration of MAA-AF555 prepared in sterile 0.5 mL microcentrifuge tubes (Fig. 2). The concentration of MAA-AF555 to be inhibited should be determined by initial

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Fig. 2 A dilution strategy for a haptenic sugar concentration series for inhibition of a fixed concentration of fluorescently labeled lectin. Starting with a stock lactose concentration of 200 mM, make the serial dilutions indicated on the left hand side to produce a final volume of 125 μL. Add 125 μL of 2 μg/mL MAA-AF555 stock to each concentration to yield 250 μL each of the final haptenic sugar concentration series on the right hand side co-incubated with a fixed 1 μg/mL concentration of MAA-AF555

titration. The concentration dilutions should be prepared half an hour in advance of the experiment and stored on ice until use. Always handle fluorescently labeled lectins in the dark. 9. Gasket slides. We use 8-well gasket slides (catalog number G2534-60015, Agilent Technologies, Inc., Santa Clara, CA, USA). 10. Metal incubation chamber assembly (one chamber per microarray slide to be incubated). We use catalog number G2534A60000 from Agilent Technologies, Inc. 11. Hybridization oven with rotating arms suitable for accommodating the incubation chamber assembly. We use the microarray hybridization oven from Agilent Technologies, Inc. (catalog number G2545A). 12. Thick walled glass staining basin. We use 120  120  50 mm staining basin with a fitted glass lid. 13. Plastic tweezers with flat, non-serrated tips. 14. Clear glass Coplin jar. 15. Sterile 50 mL polypropylene tubes.

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16. Microarray slide holders for scanning. We use Agilent slide holders for the G2505B microarray scanner (catalog number G2505-60525). 17. Microarray scanner with appropriate laser for fluorescent label. We use an Agilent G2505B DNA microarray scanner which has 532 nm (green) and 633 nm (red) lasers. 2.3 Calculation of IC50 Values from Glycomics Microarray Data

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1. Microsoft Excel 2010 (Microsoft, WA, USA). Later versions of Excel can be used. 2. GraphPad Prism v.9 (GraphPad Software, San Diego, CA, USA). Versions from 8 and later can be used.

Methods

3.1 Fluorescently Labeling MAA Lectin

1. All steps are carried out in the dark at room temperature unless otherwise indicated. A dark room with a red photographic safety light is suitable for all following steps. 2. Dissolve pure, unlabeled MAA in ultrapure water at 4 mg/mL. In an amber 2 mL tube, add 200 μL of this stock MAA solution to 200 μL of 20 mM lactose in 500 mM sodium borate, pH 8.2. The MAA is now ready for the labeling reaction at a final concentration of 2 mg/mL in 10 mM lactose in 250 mM sodium borate, pH 8.2 (see Notes 8 and 9). 3. Add 100 μL anhydrous DMSO to the 1 mg tube of Alexa Fluor® 555 succinimidyl ester dye and mix with pipette action until the dye powder is fully dissolved. 4. Add 10 μL of dissolved dye (i.e., 100 μg of Alexa Fluor® 555 succinimidyl ester dye) to the MAA in the amber tube and mix by pipette action (see Note 10). Close the cap on the amber reaction tube and incubate at 25  C in a heat block for 2 h with gentle shaking (200 rpm). 5. Approximately 10 min before the end of the labeling reaction, wash a 3 kDa molecular weight cut-off centrifugal filter with ultrapure water. This is done by pipetting 500 μL ultrapure water into the filter device. Place the device in the filtrate collection tube, close the cap, and centrifuge the tube at 14,000 rpm (20,817  g) for 10 min. Always make sure to balance tubes in the centrifuge. Retrieve the tube from the centrifuge, discard any remaining water in the filter device and filtrate collection tube, and replace the filter device in the filtrate collection tube. Close the cap of the tube when not in use (see Note 11). 6. At the end of the labeling reaction, pipette all 400 μL of the reaction mixture into the washed filter device and close the cap of the tube. Centrifuge the tube at 14,000 rpm (20,817 

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g) for 10 min. Take the filter device from the tube (being careful not to overturn the device and lose the retentate), discard the filtrate from the filtrate collection tube, and replace the filter device in the filtrate collection tube. 7. Add 400 μL of TBS to the retentate in the filter device. Close the cap of the filtrate collection tube and centrifuge the tube at 14,000 rpm (20,817  g) for 10 min. Remove the filter device from the collection tube, discard the filtrate from the collection tube, and replace the filter device in the filtrate collection tube. 8. Repeat wash step 7 four more times. The Alexa Fluor® 555 labeled MAA (MAA-AF555) is now purified and the buffer has been exchanged to TBS. 9. Recover the concentrated MAA-AF555 immediately by placing the Amicon® Ultra filter device upside down in a clean filtrate collection tube. Place the tube in the centrifuge with the cap open towards the center of the rotor. Centrifuge at 1,000 rpm (106  g) for 2 min. 10. Remove the filter device and add 100 μL TBS to the filter device with gentle mixing by pipette action. Recover the 100 μL TBS wash from the filter device in the same collection tube as described in step 9. 11. Discard the filter device and transfer the purified, pooled MAA-AF555 solution to an amber tube. Aliquots may be made if desired at this time (see Note 12). 12. To quantify the MAA-AF555 concentration, measure the absorbance of 1 μL of purified MAA-AF555 solution at 280 (A280 nm) and 555 nm (A555 nm) using a NanoDrop™ 2000 spectrophotometer (ThermoFisher Scientific). The protein concentration may be calculated using the following formula from the dye manufacturer: Protein concentration ðMÞ ¼

½A280nm  ðA555nm  0:08Þ 49, 000

where 0.08 is the correction factor to account for absorption of the dye at 280 nm (see Note 13) and 49,000 cm1 M1 is the approximate molar extinction coefficient of MAA (see Note 14). It may be necessary to dilute the sample if the sample is too concentrated. If dilution is done, the dilution factor should be accounted for in the concentration calculation. 13. A protein concentration in mg/mL or μg/mL can be calculated from the above result using the molecular mass (Mr) of the protein (e.g., Protein concentration (mg/mL) ¼ Protein concentration (M)  Mr). 14. The degree of labeling of the protein can also be calculated using the following formula from the dye manufacturer:

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moles dye per mole protein ¼

A555nm 150, 000  protein concentration ðMÞ

where 150,000 cm1 M1 is the approximate molar extinction coefficient of the Alexa Fluor® 555 dye at 555 nm provided by the manufacturer (see Notes 15 and 16). 3.2 Binding Inhibition Incubations of MAA-AF555 on Glycan Microarray Slides

1. All steps are carried out at room temperature in the dark unless otherwise indicated. A dark room with a red photographic safety light is suitable for all following steps. 2. Initially a titration of several concentrations of MAA-AF555 diluted in TBS-T on the glycan microarray must be carried out on one glycan microarray slide to select a suitable concentration of MAA-AF555 for inhibition experiments. A concentration of MAA-AF555 where no binding intensity signal is saturated and the range of binding intensities is distributed over the range of the detection of the microarray scanner is desirable (i.e., a collection of low, medium, and high intensity interactions) [10]. We recommend starting with titrating a selection of several concentrations over a range of 0.1–15 μg/ mL in TBS-T. Additional titrations may be performed if necessary. 3. Once the optimal concentration of MAA-AF555 is selected for inhibition experiments (1 μg/mL in this case), a suitable range of haptenic sugar concentrations must be determined by titration on one glycan microarray slide (Fig. 3). A concentration range demonstrating a regular distribution of binding inhibition (ideally like “steps of stairs”) which encompasses 50% of inhibition of uninhibited binding is optimal (e.g., Fig. 4b). We recommend initially titrating lectin binding inhibition by co-incubating the fixed concentration of fluorescently labeled lectin with 0.1–100 mM of the haptenic sugar to determine an optimal inhibition concentration range. In the case of 1 μg/mL MAA-AF555, a concentration range of 0.0488–100 mM lactose for inhibition was selected for generating suitable data to enable IC50 calculation. 4. The inhibition assay must be carried out in triplicate. Prepare three gasket slides in the open incubation assemblies and draw a map of the location of each lactose concentration co-incubated with MAA-AF555 as well as uninhibited MAA-AF555 on the gasket slide (Fig. 3a). Pipette 70 μL (or appropriate volume for gasket slide and glycan microarray combination used) of each lactose concentration co-incubated with MAA-AF555 as well as uninhibited MAA-AF555 into each gasket well according to the map (Fig. 3b), and repeat for three gasket slides (see Note 17).

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Fig. 3 (a) Map for pipetting samples onto gasket slide for glycan microarray incubations. (b) Photograph of pipetting 70 μL of each sample into the middle of each appropriate well on a gasket slide in an opened incubation chamber

5. Sandwich one glycan microarray slide on top of each gasket slide on which the appropriate samples have been pipetted, making sure that the printed slide of the microarray slide is in contact with the liquid pipetted into each gasket slide well and the subarrays are correctly aligned. Once the glycan microarray slide is in place on top of the gasket slide, immediately assemble the metal incubation chamber to seal the microarray slide into place. 6. Place the sealed incubation chambers in a rotating hybridization oven and incubate for 1 h at 23  C with gentle rotation (4 rpm). 7. Ten minutes before the end of the incubation period, fill a thick walled glass staining basin and a glass Coplin jar with TBS-T. 8. After incubation, remove the incubation chambers from the hybridization oven and disassemble the incubation chambers. Remove the gasket and microarray slides from the chamber. The gasket and microarray slides should be adhered together at this point and not easily parted with gentle pressure. Submerge the gasket and microarray slides in TBS-T in the glass basin, insert a flat tipped plastic tweezers between the slides at the barcode end of the microarray slide, and twist the tweezers to release the slides from one another and to immediately dilute the incubation solutions. Allow the gasket slide to fall to the

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bottom of the basin and retain a hold on the microarray slide by its edges. 9. Remove the glycan microarray slide from the basin and insert into the TBS-T-filled glass Coplin jar, being careful to never touch the printed surface of the slide at any time during handling. Wash the microarray slides in TBS-T three times by shaking the Coplin jar gently on a plate shaker for 5 min for each wash. 10. Wash the microarray slides once in TBS for 5 min and leave the slides in the Coplin jar. 11. Remove the microarray slides from the Coplin jar using the plastic tweezers and place in a sterile 50 mL tube (one microarray slide per tube). Centrifuge the tubes at 475  g (1,500 rpm) for 5 min to dry. 12. Remove the dried microarray slides from the tubes and place in a microarray slide holder suitable for the microarray scanner in use (one slide per holder). Insert the slide holders into the microarray scanner and scan at 5 μm resolution using the appropriate laser (532 nm, the green channel in this case) and photomultiplier tube (PMT) gain (20% and 90% scans were performed in this case). Save the image as a .tif file (Fig. 4a). 13. Extract the binding intensity data from the generated .tif file using microarray extraction software and the .gal file for the glycan microarray (see Note 18). Binding intensity data should be normalized across the triplicate microarray experiments to the per-subarray average total intensity and the normalized replicate data then used for calculating IC50 values. 14. Binding intensity data can be plotted as a bar chart in Excel (Fig. 4b). 3.3 Calculation of IC50 Value Using Inhibition Data from Glycan Microarray Inhibition Experiments

1. Binding intensity data in a suitable inhibitory concentration range in technical triplicate must be selected to calculate the IC50 value for inhibition of binding a particular ligand. We recommend using a concentration range including at least four concentrations that result in less than and greater than 50% of uninhibited lectin binding to a ligand (uninhibited lectin binding to a ligand is considered 100% binding), of which at least two data points are greater than the 50% inhibition and two data points are less than 50% inhibition (see Note 19). For example, lactose concentrations in the range 0.0488–100 mM co-incubated with MAA-AF555 resulted in a suitable range of binding inhibition to the neoglycoprotein ligand 3SLN-BSA (3SLNBSA) (Fig. 4). 2. The binding intensity data of the selected (normalized) data series for the particular ligand must be converted to percentage binding relative to uninhibited lectin binding, i.e., relative to

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Fig. 4 (a) Image of glycan microarray slide scan after incubation with the lactose dilution series concentrations co-incubated with 1 μg/mL MAA-AF555 as well as uninhibited MAA-AF555. (b) Bar charts of the uninhibited and lactose-inhibited MAA-AF555 binding intensities to the printed SLNBSA neoglycoprotein after scans at 20% (left) and 90% (right) PMT gain

the binding intensity of the recognition molecule in the absence of the inhibitor which is considered 100% binding. In the selected example, MAA-AF555 binding to SLNBSA in the absence of lactose was designated as 100% binding, while the binding intensities of MAA-AF555 to SLNBSA in the presence of various concentrations of lactose were converted to relative percentage binding (Fig. 5 and equation below). ðRFUinhibited =RFUuninhibited Þ  100%:

ð1Þ

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Fig. 5 Conversion of binding intensity data in the presence of various concentrations of lactose to relative percentage of uninhibited binding. Binding intensity data from lactose inhibition of MAA-AF555 binding to SLNBSA from (a) 90% and (b) 20% PMT gain scan

Equation for the conversion of binding intensity values of inhibited binding to relative percentage of uninhibited binding. RFU is relative fluorescence intensity, the binding intensity of the fluorescently labeled lectin to the printed neoglycoprotein 3. Only the inhibitory concentrations which inhibit the binding intensity of the lectin and contribute to the range of inhibition should be included in the data series for IC50 calculation. For example, the lactose concentrations 0.0488 and 0.1953 mM for the 90% scan and 0.0488 for the 20% scan (Fig. 5) did not contribute to the inhibition range, i.e., maximum inhibition was reached at the respective previous concentrations (0.78125 mM lactose for the 90% scan and 0.1953 mM for the 20% scan). The non-contributing concentration values should not be included in the IC50 value calculations (Fig. 6). 4. Open GraphPad Prism. Select “Enter or import data into a new table” in the “Data table” section of the pop-up screen (Fig. 7). In the “Options” section of the same pop-up screen, select

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Fig. 6 The data included for IC50 value calculations from lactose inhibition of MAA-AF555 binding to SLNBSA from (a) 90% and (b) 20% laser power scans

Fig. 7 The pop-up window in GraphPad Prism for entering a new data set. Image used with permission from GraphPad Software

“Numbers” for the X-axis and enter 3 replicate values in the Y-axis. Press the “Create” button (Fig. 7). 5. Copy the selected data set of triplicate relative binding percentage values into the X and Y columns of the newly opened table (Fig. 8). Enter appropriate titles for the axes.

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Fig. 8 The generated table to enter relative percentage binding from experimental data in GraphPad Prism. The “Analyze” button is indicated by the inserted red arrow. Image used with permission from GraphPad Software

6. The concentration values (X-axis values) must be transformed to log values (see Note 20). For this reason, 0 cannot be used as a value for the inhibitor concentration for the uninhibited binding intensity (i.e., 100% binding). However, a value must be entered which ideally will be well separated in the inhibition plot from the inhibition data values. Enter a value of 1 which is one decimal place lower in numerical value than the lowest inhibition concentration used. For example, in the data series presented for lactose inhibition of MAA-AF555 binding to SLNBSA, the lowest lactose concentration included was 0.1953 mM. Therefore the concentration value inserted for the uninhibited data set was 0.01 mM (Fig. 8). 7. To convert the X-axis values to log values, press the “Analyze” button in the “Analysis” section of the toolbar (Fig. 8). In the subsequent pop-up window, select the “Transform” function in the “Transform, Normalize” analysis section and press the “OK” button (Fig. 9). 8. In the next pop-up window, select “Standard functions” from the “Function List” section (Fig. 10). Then select “Transform X values using” and select “X ¼ Log(X)” from the dropdown

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Fig. 9 The pop-up window for the “Analyze Data” function in GraphPad Prism. The “Transform” function in the “Transform, Normalize” analysis section is selected. Image used with permission from GraphPad Software

menu. Check “Create a new graph of the results” and press the “OK” button (Fig. 10). The screen is then returned to the table where X-axis data has been transformed into the log values. The title of the X-axis should be updated to reflect the Log data by manually typing in a new title (Fig. 11). 9. Click the “Analyze” button again and select “Nonlinear regression (curve fit)” in the “XY analyses” section in the pop-up window (Fig. 12). Press the “OK” button. 10. In the “Model” tab of the next pop-up window, select the “Dose-response – Inhibition” set of equations and then select the “log(inhibitor) vs. response (three parameters)” equation to generate the curve (Fig. 13). Check the “Interpolate” box and select 95% as the confidence interval from the dropdown menu. Press the “OK” button (Fig. 13). The least squares fitting method is used. 11. GraphPad Prism will generate a table of results which includes the calculated IC50 value and the R2 value for the data set

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Fig. 10 The pop-up window for the “Parameters Transform” function in GraphPad Prism. The “Standard functions” from the “Function List” section is selected and the “Create a new graph of the results” box is checked in the window. Image used with permission from GraphPad Software

(Fig. 14). A R2 value of 0.9 is desirable for confidence in the results (see Note 21). If the R2 value is 0.9 but should not be 0.99. A value of 0.99 indicates that there is no mucin or protein present as mucin distorts the linear range

4. Fraction collector. We use the Bio-Rad Model 2110 fraction collector and collect eluate in drop (approximately 50 μL per drop) format. 5. Borosilicate 13  100 mm glass fraction tubes. 6. A Bio-Gel® P-6 (medium) column (2.5  50 cm) in filtered and degassed ultrapure water. Bio-Gel® P-6 medium resin is from Bio-Rad Laboratories Ltd. (catalog number 1504130). Column should be thoroughly washed with 5 CVs of buffer and blocked with 2% BSA before use. Determine flow rate (approximately 0.8 mL/min) and void volume (approximately 160 mL) before use (see Note 3). 7. Filtered and degassed ultrapure water.

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8. Sterile 50 mL tubes. 9. Sterile 1.5 mL microcentrifuge tubes. 10. Lyophilizer. 2.2 Printing Mucin Microarray Slides

1. Microarray printer. We use a SciFlexArrayer S3 (Scienion AG, Berlin, Germany) with a 20 slide printing stage and a piezoelectric 80 μm diameter coated glass nozzle (PDC 80 with Type 4 coating). 2. Nexterion® slide H microarray slides (Schott AG, Mainz, Germany). These functionalized microarray slides are supplied in packs of 25 in vacuum-packed containers and stored at 20
C. 3. Phosphate buffered saline (PBS), pH 7.4 (see Note 4). Usually 1 L of an autoclaved 10 PBS stock is made and diluted to use concentration (1) with ultrapure water just before use (100 mL of 10 PBS added to 900 mL of ultrapure water). 4. Tween® 20. 5. PBS-T: 100 mL 10 PBS and add 900 mL ultrapure water and 0.5 mL molecular biology grade Tween® 20 (0.05%) (see Note 5). 6. A selection of small volumes (e.g., 1 mL) of Tween® 20 concentrations in PBS, pH 7.4, in addition to PBS-T for diluting mucins to their optimized individual protein print and Tween® 20 concentration (see Note 5). For example, PBS with 3% Tween® 20 (3%T), 2% (2%T), 1% (1%T), 0.5% (0.5%T), and 0.2% (0.2%T). 7. A panel of purified mucins (probes) either as dry powder in 0.5 mg aliquots (from Subheading 3.1) or as frozen aliquots of 1 mg/mL in PBS, pH 7.4. Mucins must be diluted to their optimized individual print concentration with their optimized Tween 20 concentration. Dissolve dry mucins in PBS, pH 7.4, to a concentration of 1 mg/mL (based on weight). Dilute each mucin to a final volume of 20 μL in its optimized print concentration in the optimized print buffer (PBS, pH 7.4, or PBS with optimal concentration of Tween® 20) (e.g., Table 1) (see Note

Table 1 Examples of desired final print protein and Tween® 20 concentrations for individual mucins and the volumes required from stocks of 1 mg/mL mucin, PBS with varying percentages of Tween® 20, and PBS to achieve 20 μL of the desired final print concentrations Mucin print conc (mg/mL)

Print %Tween® 20

μL mucin at 1 mg/mL

0.75

0.75

15

5 (3%)

–

0.5

0.25

10

10 (0.5%)

–

0.25

0.5

5

10 (1%)

5

μL (%T)

μL PBS

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6). These mucin print dilutions must be made fresh on the day of microarray printing. Aliquot the remaining liquid volume of 1 mg/mL mucin stocks into 20–50 μL aliquots in 0.5 mL microtubes and store at 20
C until use (see Notes 7 and 8). 8. Sterile 0.5 mL microcentrifuge tubes. 9. 384-well sciSOURCEPLATE 384 PS microarray probe plate (catalog number CPG-5501-1, Scienion AG, Berlin, Germany). 10. Saturated sodium chloride (NaCl) solution. 11. Humidity chamber. We use the StainTray slide staining system with a black lid (catalog number Z670146, Sigma-Aldrich Co.) as a humidity chamber. A layer (approximately 3 mm depth) of saturated NaCl is poured at the bottom of the chamber and the chamber has raised polymer runners to keep microarray slides in place and save them from getting wet. 12. Five clear glass Coplin jars. 13. Blocking solution: 100 mM ethanolamine in 50 mM sodium borate, pH 8.0. To approximately 700 mL ultrapure water, add 3.0 g boric acid (99.5% purity, catalog number B0252, Sigma-Aldrich Co.) with constant stirring. Add several drops of 50% NaOH to the solution until the sodium borate dissolves. Add 6 mL of ethanolamine (99.0% purity, catalog number 398136-25ML, Sigma-Aldrich Co.) and then adjust the pH to 8.0 using concentrated HCl (dropwise) with constant stirring. Adjust the volume to 1 L by adding ultrapure water. Make blocking solution fresh just before use. Approximately 500 mL is required to block 20 slides. 14. 50 mL sterile polypropylene tubes. 15. Flat topped plastic tweezers. 16. Slide box to store the printed microarray slides. 17. Calcium chloride (CaCl2). 18. Resealable plastic bags.

3

Methods

3.1 Mucin Purification 3.1.1 Mucus Dissolution, Reduction, and Alkylation

1. Remove sputum aliquots (typically 0.5 mL sputum in 1.5 mL microcentrifuge tubes) from the 80
C freezer and thaw on ice. We typically process up to 8 samples at a time. 2. Once thawed, retain 2 μL of each aliquot to test for the presence of mucins via PAS blotting. To each 0.5 mL aliquot, add an equal volume of 8 M GdnCl to the sputum to give a final concentration of 4 M GdnCl. Mix the 1.5 mL tubes on a roller at room temperature until the solution is homogenous (up to

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1 h but the time is sample dependent). Remove 2 μL of the GdnCl-diluted sputum samples for PAS blotting. The solution can be stored at room temperature for up to a day until the next steps. 3. For PAS blotting, wash the PVDF membrane (pre-cut to the appropriate size for the dot blotting apparatus) in methanol by immersing the membrane in the methanol in a square plastic petri dish for 3 min. Remove the membrane from the methanol using a plastic tweezers and transfer directly to a petri dish filled with ultrapure water and allow to soak for 2 min. 4. In a separate square petri dish, soak two Whatman® chromatography papers in water for 2 min. 5. Assemble the dot blotter by placing the two wet Whatman® chromatography papers on the base of the dot blotting apparatus using gloves, layer the soaked PVDF membrane on top of the wet Whatman papers using plastic tweezers, and place the 96-well frame on top of the PVDF membrane. Screw down the 96-well frame and attach the dot blotter to the vacuum line (Fig. 2a). Add 100 μL of ultrapure water to each well of the dot blotter, open the vacuum line using the tap, and allow the liquid to be pulled through the membrane. Repeat this wash step once more and close the vacuum. 6. Pipette the retained 2 μL aliquots of the non-diluted and GdnCl-diluted sputum samples onto the membrane in separate wells of the 96-well frame of the dot blotter (Fig. 2b). The 2 μL will soak into the membrane immediately. Add 100 μL of ultrapure water on top of these samples. Include a well for the positive control of 100 μL of 1 mg/mL bovine fetuin. Allow to soak into the membrane for 10 min and then open the vacuum line and allow the liquid to be pulled through the membrane. 7. Add 100 μL of ultrapure water to each well of the dot blotter and allow the liquid to be pulled through the membrane for one wash. 8. Close the vacuum line and disassemble the dot blotter. Remove the membrane from the dot blotter using a plastic tweezers and wash the membrane in ultrapure water for 10 min in a plastic petri dish. 9. Pour off the water and add the 1% periodic acid in 3% acetic acid, making sure to completely submerge the membrane in the oxidizing solution. Cover the dish and incubate at room temperature for 30 min. 10. Wash the membrane twice for 2 min each wash in 0.1% sodium metabisulfite in 1 mM HCl.

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11. After pouring off the last 0.1% sodium metabisulfite in 1 mM HCl wash, add the Schiff’s reagent and incubate at room temperature until color appears in the mucin-positive wells (or the positive control). A purple-pink color will usually evolve within 10 min for carbohydrate-positive wells but if mucin concentrations are very low it may take up to 40 min. 12. Wash the membrane twice for 2 min each wash in 0.1% sodium metabisulfite in 1 mM HCl. 13. Rinse the membrane with methanol and allow to dry on white paper (see Note 9). Wells with a purple-pink color are PAS-positive and demonstrates the presence of carbohydrates which is taken to indicate the presence of mucins (Fig. 2c). 14. Once the GdnCl-diluted sputum samples have been verified as PAS-positive, add 1 M DTT to the PAS-positive samples to a final concentration of 10 mM DTT (e.g., add an appropriate volume of 1 M DTT stock to the sample solution to achieve a 1 in 100 dilution) and incubate for 1 h at 37
C. PAS-negative samples should not be processed any further. 15. After incubation, add iodoacetamide to final concentration of 25 mM iodoacetamide (e.g., add an appropriate volume of 250 mM iodoacetamide stock to the sample solution to achieve a 1 in 10 dilution) and incubate the sample overnight in the dark at 4
C with gentle shaking (4 rpm). 16. The dissolved, reduced and alkylated sputum samples are now stable at room temperature until further purification. 3.1.2 Isopycnic Density Gradient Centrifugation in Cesium Chloride

1. Transfer the reduced and alkylated sample to a 50 mL tube and adjust the volume to 25 mL by adding the 4 M GdnCl solution (see Note 10). Mix thoroughly on a roller at room temperature until the sample is homogenous throughout (up to 1 h). 2. Transfer the solution to a 50 mL glass beaker, add a stir bar, and stir the solution on a magnetic stir plate. Remove 1 mL of solution and weigh by placing the solution in a pre-weighed 1.5 mL tube, capping the tube and weighing it, and subtracting the weight of the tube alone. This will give the density of the solution in g/mL. Replace the 1 mL of solution into the bulk solution in the beaker after weighing. 3. Add CsCl in 5 g aliquots or less to the solution and dissolve fully by gentle stirring for approximately 10 min (see Notes 11 and 12). Weigh the solution after each addition and dissolution of CsCl as described in step 2, making sure to replace each time the 1 mL of solution that was removed for weighing back in to the bulk solution. Continue to add CsCl until the density of solution reaches 1.4 g/mL (+/0.005 g) (see Note 13).

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4. Transfer the solution back to the 50 mL tube and adjust the volume to 30 mL by adding 1.4 g/mL CsCl in 4 M GdnCl. 5. Transfer the solution to a disposable 30 mL ultracentrifuge tube (see Notes 14 and 15) (Fig. 3a). 6. Centrifuge the ultracentrifuge tubes at 65,000 rpm (approx. 425,000  g) for 18 h at 10
C. 7. Gently remove the ultracentrifuge tubes from the rotor and unpack the CsCl gradient by removing 1 mL aliquots from the top of the solution using a 1 mL pipette tip. Place each 1 mL aliquot or fraction into a separate 1.5 mL microcentrifuge tube numbered in order of removal (i.e., fraction 1 for the first aliquot removed, fraction 2 for the second aliquot, and so on). Approximately 30 fractions will be collected in total (see Note 16). 8. Weigh 0.5 mL of each density gradient centrifugation fraction, making sure to replace the volume in each fraction tube after weighing. Multiply the resulting solution weight by 2 to calculate the density in g/mL of each fraction. 9. Plot the density of each fraction against the fraction number (Fig. 3b). The density of fractions should increase with fraction number (i.e., from the top to the bottom of the ultracentrifuge tube). Mucins are found in fractions 1.35–1.45 g/mL. 10. Each density gradient centrifugation fraction must be tested for PAS positivity in a dot blot (use 50 μL of each fraction for steps 3–13 in Subheading 3.1 above) (see Note 17). 11. Pool the fractions which are PAS-positive and have a density of 1.35–1.45 g/mL. These fractions contain mucin. Mucins must be further purified and desalted by gel permeation chromatography. 3.1.3 Mucin Purification and Desalting by Gel Permeation Chromatography

1. Remove the cap from the Sepharose CL 4B column and open the tap at the bottom of the column (Fig. 4). Allow the eluent to drain to the level of the top of the resin bed at the top of the column. Before allowing the resin to dry and leaving the tap open, apply approximately 6 mL of the pooled mucincontaining CsCl fractions (sample; from Subheading 3.1.2) dropwise to the top of the resin bed without disturbing the top of the resin bed (see Note 18). 2. After the applied sample has absorbed into the resin and there is no liquid remaining at the top of the column (but the resin has not been allowed to start to dry), apply approximately 6 mL of 50 mM Tris/100 mM KCl, pH 7.5, dropwise using a glass Pasteur pipette without disturbing the top of the resin bed. 3. Allow the buffer to absorb into the resin until there is no liquid remaining at the top of the column but do not allow the resin

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Fig. 4 Example of a size exclusion column set up with a fraction collector and eluent reservoir. Eluent reservoir performs optimally when placed higher than the top of the column

to dry. Apply approximately 6 mL of 50 mM Tris/100 mM KCl, pH 7.5, dropwise using a glass Pasteur pipette without disturbing the top of the column bed. Replace the cap of the column and restore the eluent flow from the reservoir. Start collecting eluate from the bottom of the column, collecting 100 drops per fraction in glass fraction tubes (approximately 4 mL per fraction). 4. After collection of 80 fractions, test all fractions for PAS reaction using 100 μL of each fraction in a dot blot (following steps 3–13 in Subheading 3.1.1 above) (see Note 17). 5. Pool the PAS-positive fractions together (Fig. 5a). Split the total volume between 50 mL polypropylene tubes (approximately 20 mL per tube). Pierce holes in the tube caps and cover the tube mouths with low shed Kim wipes before screwing the pierced lids back on the tubes. Freeze the liquid in the 50 mL

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Fig. 5 (a) Example of a PAS dot blot carried out on fractions from a partially purified sputum sample following size exclusion chromatography on a Sepharose CL 4B column. Mucins are present in the PAS-positive fractions eluted in the void volume (fractions 20 to 52 in this example). (b) Example of a PAS dot blot carried out on fractions from a purified sputum sample following desalting on a Bio-Gel® P-6 column

tubes overnight in a 80
C freezer. The tubes should be tilted slightly so that the liquid freezes at an angle to increase surface area for faster lyophilization. 6. Place the frozen tubes in lyophilizer jars and lyophilize the liquid to complete dryness. Lyophilization can take up to 3 days. 7. When the samples have been fully lyophilized, dissolve and pool all of the remaining lyophilized powder into the lowest volume of degassed ultrapure water that it takes to dissolve the powder completely (approximately 5 mL). 8. The samples must then be desalted on a Bio-Gel® P-6 column eluted with degassed ultrapure water. Apply the 5 mL of sample from step 7 to the Bio-Gel® P-6 column as described in step 1 and allow to absorb into the resin (Fig. 4). 9. Apply 5 mL of degassed ultrapure water to the resin as per step 2 and then another 5 mL as per step 3. Replace the cap of the

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column and restore the eluent flow from the ultrapure water reservoir. Start collecting eluate from the bottom of the column, collecting 50 drops per fraction in glass fraction tubes (approximately 2 mL per fraction). 10. After collection of 80 fractions, test all fractions for PAS reaction using 100 μL of each fraction in a dot blot (following steps 3–13 in Subheading 3.1.1 above) (see Note 17). Pool the PAS-positive fractions together (Fig. 5b). 11. Freeze and lyophilize the pooled desalted mucin-containing fractions as described in steps 5 and 6. Lyophilization will take approximately 1–2 days. 12. Weigh the now dry mucin powder by weighing the tube with the mucin powder and subtracting the weight of an identical empty tube or the same clean and dry tube after removal of the mucin powder. 13. Dissolve the dry mucin in the minimum (known) volume of degassed ultrapure water. The mucin may be desalted again on the Bio-Gel® P-6 column (steps 7–11) (see Note 19) or aliquoted for lyophilization for storage. 14. For storage, aliquot the dissolved mucin between 1.5 mL tubes such that each tube contains 0.5 mg of mucin by volume. Freeze the tubes and lyophilize the liquid in the tubes to complete dryness. Seal the tubes containing the dry mucin of known weight with parafilm and store at 20
C until use. 3.2 Printing Mucin Microarray Slides

1. Complete the microarray printer startup protocols as per manufacturer’s instructions to attach the coated glass nozzle to the pump, clean the printer lines, and adjust the piezoelectric nozzle voltage and frequency to create a stable print drop using the system liquid (water). Set the room temperature to 20
C and microarray printer humidity to 62+/2% (see Note 20). 2. Remove a pack of vacuum sealed Nexterion® slide H microarray slides from the freezer and thaw on the bench 30 min before use. 3. Remove aliquots of lyophilized mucins from the freezer and allow to equilibrate to room temperature and thaw any frozen 1 mg/mL mucin aliquots on ice. Centrifuge the lyophilized mucins (1000  g, 2 min) to bring the mucin powder to the bottom of the tube. Add the appropriate volume of PBS, pH 7.4, to the lyophilized mucins for a final concentration of 1 mg/mL. Mix the mucin solutions by vortexing and centrifuge the aliquots at 1000  g for 1 min to bring the liquid to the bottom of the tubes. Remove the microtubes carefully from the centrifuge and place in a plastic rack in print order. Leave the plastic rack on ice.

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Fig. 6 Example of mucins printed at different concentrations in different print buffers to optimize feature formation. (a) Equine duodenum mucin printed at indicated concentrations in PBS, pH 7.4, supplemented with 0.01% Tween® 20 and incubated with 10 μg/mL MAA-TRITC. The 0.3 mg/mL concentration was selected for mucin microarray printing [3]. (b) Bovine endometrium mucin printed at indicated concentrations in PBS, pH 7.4, supplemented with 0.025% Tween® 20 and incubated with 10 μg/mL UEA-I-TRITC. The 0.4 mg/mL concentration was selected for mucin microarray printing [3]

4. Make 20 μL dilutions of the 1 mg/mL mucins to the required print concentration and print buffer (e.g., Table 1). All mucins must have been previously individually optimized for optimal print concentration and print detergent concentration (Fig. 6). Place the diluted mucins in a plastic rack in print order and keep on ice. 5. Pipette the entire 20 μL of the mucins diluted to their optimized print concentration and in their optimized print buffer into the microarray probe plate in print order. Place the lid on the probe plate and centrifuge the plate at 1500 rpm (168  g) for 2 min to pull the liquid to the bottom of each well and remove any air bubbles (see Note 21). Carefully remove the probe plate from the centrifuge, making sure not to disturb the liquid in the wells, and insert the probe plate onto the plate chiller (set at 10
C) in the microarray printer housing (see Note 22). 6. Open the sealed pack of microarray slides and fit 20 microarray slides into the individual places on the printer stage (see Notes 23–25). Make sure to place the slides with functionalized surface facing up for printing and barcode orientated correctly. Once the slides are loaded, remove the lid from the probe printing plate and close the printer housing door. 7. Following the microarray printer manufacturer’s instructions for the operating software, load the desired .gal file for printing and select the locations of microarray slides to be printed on the printer stage. Start the print run (see Note 26). 8. After the completed print run, remove the printed mucin microarray slides from the printer stage and place the microarray slides in the humidity chamber with printed side facing upwards. Place the lid on the chamber and incubate the slides

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in the humidity chamber overnight at room temperature (20
C) to facilitate probe conjugation. Do not allow the microarray slides to get wet. 9. Block any remaining active functional groups on the functionalized microarray slide surface by placing the microarray slides in dry clean glass Coplin jars, four slides to a jar and incubating in blocking buffer for 1 h at room temperature without shaking. Make sure the microarray slides are placed barcode side at the top of the Coplin jar to allow handling the slides without damaging the printed mucins and that the printed sides of the microarray slides do not touch the sides of the Coplin jar or each other. 10. Wash the microarray slides in PBS-T three times after blocking, 5 min per wash with gentle shaking on a plate shaker, followed by a final wash in PBS to remove any detergent from the slide. Do not pour off the last PBS wash. The slides should never be allowed to dry after blocking until they are dried evenly by centrifugation. 11. Using a plastic tweezers to grip the microarray slides at the barcode, transfer the blocked and washed microarray slides to 50 mL polypropylene tubes, one slide per tube, orientating the barcode side of the microarray slides towards the top of the tube. Screw the cap onto the tube as soon as each microarray slide is placed in the tube to avoid uneven drying on the slide surface. Centrifuge the 50 mL tubes at 1,500 rpm (475  g) for 5 min (see Note 27) and remove the tubes from the centrifuge carefully to avoid re-wetting the evenly dried microarray slides. 12. Remove the mucin microarray slides from the tubes using a plastic tweezers to grip the barcode of the microarray slide and only handle the edges of the slide. Place the microarray slides into a slide box and store with desiccant at 4
C in a resealable plastic bag (see Note 28). Use the mucin microarrays within 6 weeks of manufacture (see Note 29). 13. One slide from each mucin microarray print batch should be validated for printing and intact feature presentation before use by incubating with a panel of fluorescently labeled lectins with specificity for mucin-type glycosylation (e.g., Table 2) (see Note 30).

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Table 2 A panel of lectins suitable for use for printing validation of mucin microarrays, their sources, and carbohydrate binding specificities Lectin

Source

Carbohydrate binding specificity

Con A

Canavalia ensiformis

Man in high-mannose type, hybrid type, and biantennary complex type N-glycans

WFA

Wisteria floribunda

GalNAc/sulfated GalNAc

AIA

Artocarpus integrifolia

(Neu5Ac)gal-β-(1 ! 3)-GalNAc-α-O-S/T (T-antigen)

PNA

Arachis hypogaea

Gal-β-(1,3)-GalNAc

UEA-I

Ulex europaeus

Fuc-α-(1 ! 2)-Gal-R

MAA

Maackia amurensis

Neu5Ac/Gc-α-(2 ! 3)-Gal-β-(1 ! 4)-GlcNAc-β-(1 ! R

SNA-I

Sambucus nigra

Neu5Ac-α-(2 ! 6)-Gal(NAc)-R

4

Notes 1. Guanidinium chloride is also known as guanidine hydrochloride and can be abbreviated as GdnCl, GuHCl, or GdmCl. 2. We have used sodium bisulfite as an alternative to sodium metabisulfite and observed similar results. 3. Mucins elute in the void volume. 4. The correct print buffer pH is critical for successful conjugation of mucins and glycoproteins to the functionalized microarray surface. 5. Tween® 20 is moderately viscous. Maintain a very slow draw when pipetting and dispensing the detergent or use a positive displacement pipette. Invert the PBS-T to mix after addition of the detergent and allow at least 15 min for the detergent to dissolve in the PBS before use. 6. Do not make a mucin print dilution in PBS with Tween® 20 in a volume in excess of that needed for the microarray print (i.e., greater volume than 20 μL) and do not store mucins diluted in Tween® 20 solutions. We note decreased quality of print performance from mucins diluted in Tween® 20 solutions stored frozen or for several days at 20
C. 7. Mucin stocks can be made, aliquoted (e.g., 20–50 μL aliquots in 500 μL microtubes) and frozen at 20
C. Aliquots stored at 20
C can be removed and thawed on ice just before use and diluted to the required print concentration and print buffer. 8. Minimal quantities of frozen aliquots of mucins diluted in PBS, pH 7.4, should be stored frozen at 20
C and dry (lyophilized) mucin storage is preferable. We have noted a degradation in print quality of diluted mucin stocks stored at 20
C for over 12 months.

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9. Do not use colored paper. The color will soak into the membrane while it is drying and obscure the PAS-positive reactions. 10. This protocol has been optimized for isolation of mucins from sputum so 4 M GdnCl is used. However for isolation of mucins from other mucus sources, 6 M GdnCl is more typical. 11. Stir the solution for 10–15 min after each addition of CsCl. The CsCl must be fully dissolved before weighing the solution. 12. Gentle stirring will ensure that bubbles are not generated in the solution. Bubbles are undesirable as they affect the weight of solution. 13. If too much CsCl is added (i.e., if the density is over 1.4 g/ mL), add sufficient drops of 4 M GdnCl to adjust the weight back to 1.4 g/mL. 14. It is very important that the ultracentrifuge tubes are balanced between samples. Weigh the ultracentrifuge tubes after adding the solution. The sets of ultracentrifuge tubes with their caps must be within 0.01 g of one another. Weigh and adjust with either 4 M GdnCl or 1.4 g/mL CsCl in 4 M GdnCl if necessary. 15. Make sure to add the solution to the ultracentrifuge tubes by pouring slowing down the side of the tube to avoid generating or trapping air bubbles. Air bubbles will affect the weight of solution in the ultracentrifuge tubes. 16. The solution near the bottom of the ultracentrifuge tube can be difficult to access with the pipette tip. In this case, the top of the ultracentrifuge tube can be cut off using a heated scalpel blade to access the lower fractions. 17. Fractions may be alternatively tested for PAS reactivity using the PAS microtiter plate assay [11] which requires only 25 μL of sample. 18. Applied sample volume should not be more than 2.5% of the total column volume. 19. If the mucin weight is too high (e.g., >5 mg from 0.5 mL sputum), the mucin should be desalted again to ensure total elimination of the salt. Typically 1–2 rounds of desalting on the Bio-Gel® P-6 column is sufficient. 20. Maintaining the optimal humidity is critical for mucin microarray printing. If the humidity is too low, the mucin spot will dry too quickly, the resulting feature will be distorted, and the data may not be extractable. 21. Make sure to include a balance plate in the centrifuge. 22. Use of a probe plate chiller will ensure minimal evaporation of the mucins from the plate during the print run (typically up to 9.5 h). We recommend printing two batches of mucin microarray slides back-to-back from the same probe plate to maximize output from the mucin aliquots.

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23. Only ever handle the microarray slides by their sides, both before and after printing. Never touch the microarray slide surfaces to avoid leaving permanent marks and deactivating functional coating. 24. Load functionalized microarray slides only when ready to print. Do not leave functionalized slides to sit for long periods as the functional groups will be deactivated by hydrolysis from environmental humidity. 25. As functionalized microarray slides are supplied in packs of 25, the remaining 5 slides can be resealed with desiccant and stored at 20
C for later use. Vacuum packed resealed slides can be stored until expiration of slides but slides resealed without vacuum packing should be used within 48 h. 26. We print two drops per feature (i.e., approximately 900 pL probe per feature) which results in circular features of approximately 230 μm diameter. Our typical print run of 20 microarray slides of 52 probes in 6 replicate features each per subarray and 8 replicate subarrays per slide takes approximately 9.5 h. 27. Balance the tubes in the centrifuge. 28. We usually use CaCl2 in a polypropylene tube as a desiccant. The tube cap is pierced to allow the moisture to be trapped by the CaCl2 and the mouth of the tube is covered with a thin low shed tissue (e.g., Kim wipe) to keep the CaCl2 powder in the tube. 29. Natural mucin microarrays should be used within 6 weeks of construction. We have observed a slight decrease in signal intensity from lectin binding experiments after 2 months which may indicate degradation of the mucin glycosylation or mucin. 30. Lectins with various fluorescent labels suitable for use with glycan microarrays are commercially available from many international reagent companies including EY Laboratories Inc. (San Mateo, CA, USA), Sigma-Aldrich Co., Vector Laboratories (Burlingame, CA, USA), Elicityl (Crolles, France), and Thermo Fisher Scientific.

Acknowledgments MK thanks the Royal Society of Chemistry (RSC) Analytical Chemistry Trust Fund (ACTF) for the 2018 RSC ACTF Fellowship award.

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References 1. Wheeler KM, Ca´rcamo-Oyarce G, Turner BS, Dellos-Nolan S, Co JY, Lehoux S, Cummings RD, Wozniak DJ, Ribbeck K (2019) Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection. Nat Microbiol 4: 2146–2154 2. Jin C, Kenny DT, Skoog EC, Padra M, Adamczyk B, Vitizerva V, Thorell A, Venkatakrishnan V, Linde´n SK, Karlsson NG (2017) Structural diversity of human gastric mucin glycans. Mol Cell Proteomics 16: 743–758 3. Kilcoyne M, Gerlach JQ, Gough R, Gallagher ME, Kane M, Carrington SD, Joshi L (2012) Construction of a natural mucin microarray and interrogation for biologically relevant glyco-epitopes. Anal Chem 84:3330–3338 4. Flannery A, Gerlach JQ, Joshi L, Kilcoyne M (2015) Assessing bacterial interactions using carbohydrate-based microarrays. Microarrays 4:690–713 5. Earley H, Lennon G, Balfe A, Kilcoyne M, Clyne M, Joshi L, Carrington S, Martin ST, Coffey JC, Winter DC, O’Connell PR (2015) A preliminary study examining the binding capacity of Akkermansia muciniphila and Desulfovibrio spp. to colonic mucin in health and ulcerative colitis. PLoS One 10:e0135280 6. Le Berre M, Gerlach JQ, Loughrey C, Creavin A, Gallagher M, Carrington SD, Joshi L, Kilcoyne M (2021) Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators. Mol Omics 17:338–346

7. Dunne C, Naughton J, Duggan G, Loughrey C, Kilcoyne M, Joshi L, Carrington S, Earley H, Backert S, RobbeMasselot C, May FEB, Clyne M (2018) Binding of Helicobacter pylori to human gastric mucins correlates with binding of TFF1. Microorganisms 6:44 8. Houeix B, Synowsky S, Cairns MT, Kane M, Joshi L, Kilcoyne M (2019) Identification of putative adhesins and carbohydrate ligands of Lactobacillus paracasei using a combinatorial in silico and glycomics microarray profiling approach. Integr Biol 11:315–329 9. Singh AK, Nguyen TH, Vidovszky MZ, Harrach B, Benko¨ M, Kirwan A, Joshi L, ˜ ada FJ, Jime´nezKilcoyne M, Berbis MA, Can Barbero J, Mene´ndez M, Wilson SS, Bromme BA, Smith JA, van Raaij MJ (2018) Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre. J Gen Virol 99:1494–1508 ˜ o K, Dolan B, Reid C, 10. Naughton JA, Marin Gough R, Gallagher ME, Kilcoyne M, Gerlach JQ, Joshi L, Rudd P, Carrington S, Bourke B, Clyne M (2013) Divergent mechanisms of interaction of Helicobacter pylori and Campylobacter jejuni with mucus and mucins. Infect Immun 81:2838–2850 11. Kilcoyne M, Gerlach JQ, Farrell MP, Bhavanandan VP, Joshi L (2011) Periodic acid–Schiff’s reagent assay for carbohydrates in a microtiter plate format. Anal Biochem 416: 18–26

Chapter 9 Bacterial Microarrays for Examining Bacterial Glycosignatures and Recognition by Host Lectins Marı´a Asuncio´n Campanero-Rhodes and Dolores Solı´s Abstract The surface of bacteria displays diverse carbohydrate structures that may significantly differ among bacteria with the same cell wall architecture and even among strains of a given bacterial species. These structures are often recognized by lectins of the innate immune system for triggering defense responses, although some bacterial pathogens exploit recognition by host lectins for favoring infection. Bacterial microarrays are a useful tool for profiling accessible bacterial surface glycans and for exploring their recognition by innate immune lectins. The use of array-printed bacterial cells enables evaluation of the recognition of the glycan epitopes in their natural presentation, i.e., preserving their real density and accessibility. Glycosylation patterns of bacterial surfaces can be examined by testing the binding to the bacterial arrays of a panel of lectins with known carbohydrate-binding preferences, and the recognition of surface glycans by innate immune lectins can easily be assessed using similar binding assays. Key words Bacterial glycans, Glycoprofiling, Lectins, Bacteria–lectin interactions, Innate immune system, Microarrays

1

Introduction Bacterial surfaces are covered with a diversity of carbohydrate structures [1] that may substantially differ from one species to another. Many bacteria can be classified into two main categories, i.e., Gram-negative and Gram-positive, which are distinguished by different cell wall architectures and the presence of specific surface glycans. In particular, Gram-negative bacteria display membraneanchored lipopolysaccharides that may or may not contain (in the latter case referred to as lipooligosaccharides) a chain extension built by repeating saccharide units, commonly designated as O-antigen. In contrast, Gram-positive bacteria are characterized by presenting a thick peptidoglycan layer over the cell membrane, and usually display teichoic acids anchored to the membrane or covalently bound to the peptidoglycan. Both Gram-negative and -positive bacteria may also present capsular polysaccharides and cell

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_9, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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surface glycoproteins. Still, the precise structure of all these glycans may significantly differ among bacteria with the same cell surface architecture, and even among strains of a given bacterial species. Moreover, bacterial surfaces may suffer alterations due to adaptation of the bacteria to changing environments, what may have consequences not only on the relative abundance and presentation of the surface glycans but also on their specific structure. Lectin microarrays have been used to examine bacterial glycans directly on the cell surface, facilitating bacteria identification and differentiation among strains, and to spot variations associated with changes in culture conditions. For example, dissimilar binding patterns were observed for two Campylobacter jejuni strains cultured at two different temperatures, what could be indicative of a decreased expression or alteration of specific glycan structures [2]. Different host receptors work co-ordinately as sentinels of host defense against bacterial infections by detecting “non-self” bacterial glycans and triggering defense responses [3–5]. However, some bacteria display self-like glycans with the aim of camouflaging from the host, or even for exploiting recognition by host lectins for downregulating the immune response or for promoting attachment through lectin bridging of bacterial and host cells. Indeed, the virulence degree of bacterial serotypes with distinct surface glycans appears to correlate with the recognition, or lack of recognition, of such structures by lectins of the innate immune system. The use of microarrays incorporating natural or synthetic bacterial carbohydrate structures has greatly facilitated the identification of potential ligands for these lectins and dissection of the recognized glycotopes [6–8]. Still, the usefulness of these microarrays is limited by the library of probes printed in the array. If a lectin targets a specific structure that is not included in the array, the analysis may not yield any meaningful binding signal. An example of strict binding specificity was the exclusive recognition by the innate immune receptor Dectin-1 of long β-(1,3)-linked glucooligosaccharides in a microarray containing a collection of 187 diverse carbohydrate structures [9]. On the other hand, the clustered presentation of the printed glycans may substantially differ from their natural density and accessibility on the bacterial surface. Therefore, the possibility that structures recognized in the arrays might not play a significant role in the lectin–bacterial cell interplay does exist. Therefore, besides assessing lectin recognition of isolated bacterial glycans for identification of ligand candidates, analysis of lectin binding to the entire bacteria is fundamental. To this aim, we developed a microarray approach based on the generation of bacterial microarrays [10, 11]. The usefulness of bacterial microarrays for exploring surface glycans and their recognition by host receptors was demonstrated using two model respiratory pathogenic bacteria displaying different carbohydrate structures, i.e., Klebsiella pneumoniae, a

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capsulated bacterium presenting an O-antigen-containing lipopolysaccharide, and non-typeable Haemophilus influenzae (NTHi), which is non-capsulated and presents a lipooligosaccharide without O-antigen extension [10, 12]. The glycosylation patterns of the bacterial surfaces were examined by testing the binding of a panel of lectins with known carbohydrate-binding preferences, and the specificity of the binding was assessed by carrying out parallel assays in the presence of suitable lectin haptens. The analysis yielded a lectin-binding fingerprint for each bacterium, revealing the presence of distinct carbohydrate epitopes. Identification of specific structures recognized by the lectins could be achieved by comparing the binding patterns of wild-type and mutant strains lacking selected surface components. In this way, the O-antigen was found to be the primary structure recognized in K. pneumoniae strain Kp52145 by the galactose-specific lectins tested [10], while in NTHi strain 375 the lipooligosaccharide served as ligand for certain galactose- and sialic acid-binding lectins [12, 13]. More important, the analysis revealed the accessibility of carbohydrate epitopes that could be recognized by lectins of the innate immune system with the appropriate carbohydrate-binding specificity. Therefore, the binding of selected innate immune lectins was next evaluated using similar binding and competition assays. Following this strategy, the first experimental evidence for direct binding to several NTHi clinical isolates of surfactant protein D, a lectin secreted into the alveolar fluid and known to recognize a wide range of respiratory pathogens [3], was obtained [12]. Thus, the microarray approach provided information on the glycosignatures of the tested bacteria and helped to unveil recognition by relevant host lectins. The approach involves direct printing of fixed bacteria (see Note 1) onto microarray slides and subsequent lectin binding assays. Key steps include: (1) Labeling of bacteria with a membrane-permeable fluorescent dye. This enables to monitor location and quantity of bacteria once printed in the arrays, thereby facilitating the control of the quality, reproducibility, and stability of the bacterial microarrays. (2) Printing of fluorescently labeled bacteria onto nitrocellulose-coated glass slides. This surface showed the best performance in terms of quantity and fluorescence intensity of printed bacteria when compared to other immobilization alternatives, as covalent immobilization on epoxy-coated slides or N-hydroxysuccinimide-activated 3-D polymer surfaces. (3) Binding assays with lectins of known carbohydrate-binding preferences (for examining the availability of carbohydrate epitopes on the bacterial surface) or with lectins of the innate immune system. The biotin/ streptavidin system is used for monitoring the binding of the tested targets. (4) Quantitation and critical evaluation of binding signals. Depending on the intensity of the signals, this may be a crucial step for differentiating relevant from non-relevant results.

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Materials

2.1 Fluorescent Labeling of Bacteria

1. UV-Vis spectrophotometer. 2. Bench centrifuge. 3. TBS: 10 mM Tris–HCl, pH 7.8, 0.15 M NaCl. 4. SYTO® 13 (Molecular Probes) green-fluorescent nucleic acid stain (see Note 2), 5 mM stock solution in DMSO. 5. Spectrofluorimeter.

2.2 Preparation of Microarrays

1. Bacteria printing buffer: 70.5% glycerol and 0.09% (v/v) Triton X-100, previously filtered with a 0.2 μm filter. 2. Microwell plates: Microseal® 384-well PCR polypropylene plates with cone-shaped wells. 3. Plate sealing film. 4. Centrifuge equipped with a plate rotor (Eppendorf™ centrifuge 5804 with Deepwell Plate Rotor A-2-DWP). 5. 16-pad nitrocellulose-coated glass slides (Grace Bio-Labs ONCYTE NOVA or SUPERNOVA slides) (Fig. 1). 6. Non-contact robotic microarrayer (Sprint, Arrayjet Ltd), equipped with a temperature and humidity control system (JetMosphere™, Arrayjet Ltd) (see Note 3). 7. Arrayer washing buffer: 47% glycerol and 0.06% (v/v) Triton X-100, filtered with a 0.2 μm filter. 8. Cy3 solution: 1 mg/mL of Cy3 mono NHS ester (GE Healthcare) in Milli-Q water (see Note 4). 9. Control (glyco)proteins: 1 mg/mL solutions of fetuin, asialofetuin, ribonuclease B, and ribonuclease A (see Note 5) in arrayer washing buffer containing 1 μL of Cy3 solution per mL.

2.3 Microarray Binding Assays

1. Slide module (Proplate, Grace Bio-Labs) (Fig. 1, see Note 6). 2. Washing buffer: TBS, PBS (5 mM sodium phosphate, pH 7.2, 0.2 M NaCl), or any other suitable buffer for the lectin to be tested (see Notes 7 and 8). 3. Blocking buffer: 0.25% (v/v) Tween 20 (see Note 9) in the selected buffer. 4. Overlay buffer: 0.1% (v/v) Tween 20 (see Note 9) in the selected buffer. When required, it may contain divalent cations (e.g., Ca2+ or Mg2+). 5. Lectins for glycoprofiling: panel of biotinylated lectins with complementary carbohydrate-binding specificities (see Note 7). Recommended lectins for initial glycoprofiling analyses are listed in Table 1.

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Fig. 1 Assembly of a 16-pad nitrocellulose-coated slide and the Proplate slide module. The module is placed over a printed slide and the ensemble is fixed with two lateral clips, creating 16 incubation chambers in which up to 16 independent binding assays can be performed simultaneously

6. Innate immune lectins: unlabeled lectins or biotinylated, His-tagged, or Fc-fusion lectins (see Note 8). 7. Lectin haptens: usually mono- or disaccharides (see Notes 7 and 8). 8. Biotin-labeled antibodies: anti-lectin, -His tag, -Fc antibodies (see Note 10). 9. AlexaFluor-647 (AF647)-labeled streptavidin (Invitrogen): 2 mg/mL solution in PBS, 5 mM azide (as provided by the manufacturer). 2.4 Microarray Scanning and Data Analysis

1. Microarray scanner (GenePix Autoloader 4200AL or later versions, Axon Instruments). 2. GenePix Pro 6.1 scanner software (or later versions). 3. Complementary software (Microsoft Excel, Microcal Origin, GraphPad, or similar).

3

Methods

3.1 Fluorescent Labeling of Bacteria

1. Centrifuge suspensions of fixed bacteria 2 min at 15,000
g or 4 min at 9000
g and resuspend in TBS. 2. Adjust bacterial suspensions to OD600 ¼ 1.0, add 1 μL of SYTO® 13 stock solution per mL of suspension, mix thoroughly, and incubate for 5 min in the dark. 3. Following labeling, wash bacteria once with TBS, adjust again bacterial suspensions to OD600 ¼ 1.0, and register the fluorescence emission spectra from 500 to 600 nm upon excitation at 488 nm (see Note 11).

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Table 1 Recommended lectins for initial glycoprofiling analyses Sugar-binding Monosaccharide preferences

Lectin

Abbreviation Source

Amaryllis lectin, Hippeastrum hybrid lectin

AL, HHL

Hippeastrum hybrid bulbs

Man

Terminal and internal α(1,3)- or α(1,6)linked Man residues. Not Glc or GlcNAc

Concanavalin A

ConA

Canavalia ensiformis seeds

Man/Glc

α-Methylmannopyranoside > α-Man > α-Glc > α-GlcNAc

Pisum sativum agglutinin

PSA

Pisum sativum seeds

Man/Glc

α-Man or -Glc, α-methylmannopyranoside

Wheat germ agglutinin

WGA

Triticum vulgaris

GlcNAc

[GlcNAc]3, [GlcNAc]2, GlcNAc. It may bind Neu5Ac, but not Neu5Gc

Griffonia simplicifolia lectin II

GSL-II

Griffonia simplicifolia seeds

GlcNAc

Terminal α- or β-GlcNAc residues

Datura stramonium lectin

DSL

Datura stramonium seeds

GlcNAc

β(1,4)-linked GlcNAc oligomers: Chitotriose > chitobiose > GlcNAc. Also LacNAc and LacNAc oligomers

Peanut agglutinin

PNA

Arachis hypogaea peanuts Gal

Galβ(1,3)GalNAc (T-antigen), Lac

Ricinus communis agglutinin

RCA-I, RCA120

Ricinus communis seeds

Gal

Terminal β-Gal. Galβ (1,4)Glc >> Galβ (1,3)Glc. GalNAc is a very poor inhibitor

Soybean agglutinin

SBA

Glycine max seeds

GalNAc/Gal

Terminal α- or β-linked GalNAc and to a lesser extent Gal residues

Wisteria floribunda agglutinin

WFA

Wisteria floribunda seeds GalNAc

GalNAc α- or β-linked to position 3 or 6 of Gal

Dolichos biflorus agglutinin

DBA

Dolichos biflorus seeds

GalNAc

Terminal α-GalNAc

Aleuria aurantia mushrooms

Fuc

Fuc α(1,6)-linked to GlcNAc or α(1,3)-

Aleuria aurantia AAL lectin

(continued)

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Table 1 (continued)

Lectin

Abbreviation Source

Sugar-binding Monosaccharide preferences linked to LacNAc related structures

Maackia amurensis lectin II

Maackia amurensis seeds Neu5Ac

Neu5Acα(2,3)Galβ (1,3)GalNAc

Sambucus nigra SNL, EBL lectin, elderberry bark lectin

Sambucus nigra bark

Neu5Ac

Neu5Acα(2,6)Gal/ GalNAc

Limulus polyphemus agglutinin

Limulus polyphemus hemolymph

Neu5Ac, Neu5Gc

Neu5Ac, Neu5Gc, and GlcNAc. D-glucuronic acid to some degree

MAL-II

LPA

Man mannose, Glc glucose, GlcNAc N-acetylglucosamine, Gal galactose, GalNAc N-acetylgalactosamine, Fuc fucose, Neu5Ac N-acetylneuraminic acid, Neu5Gc N-glycolylneuraminic acid, LacNAc N-acetyllactosamine

3.2 Preparation of Microarrays (See Note 12)

1. Centrifuge suspensions of SYTO® 13-labeled bacteria in TBS adjusted to OD600 ¼ 1.0 (2 min at 15,000
g or 4 min at 9000
g). Remove two-thirds of the volume from the supernatant and replace it with the same volume of bacteria printing buffer. Mix until a homogeneous suspension is obtained (see Note 13). 2. Prepare serial dilutions of bacteria in arrayer washing buffer, covering the desired dose-response range (commonly four levels from OD600 ¼ 1.0 to 0.1). These dilutions can be stored at 20  C until used. 3. Prepare serial dilutions of control (glyco)proteins, from 1 to 0.03 mg/mL, in arrayer washing buffer containing 1 μL of Cy3 solution per mL. 4. Transfer 10 μL of each sample to a 384-microwell plate following an arrangement leading to an easily recognizable layout in the printed slides. Seal the microplate with film to avoid sample contamination, centrifuge the microplate at 200
g for 1 min (see Note 14), and proceed immediately with printing. 5. Set the temperature and humidity of the equipment at 20  C and 50%, respectively (laboratory temperature controlled at 22  C), and place microplates and slides in their respective holders within the arrayer (see Note 15). 6. Print serial dilutions of bacteria and control (glyco)proteins in duplicates, triplicates, or quadruplicates, depending on the

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desired layout. Replicate microarrays of 216 spots (in a matrix of 12
18 spots) can be easily printed within each pad of a 16-pad slide. Up to 20 slides can be printed in a single run. 7. Let the slides dry overnight in a dark place. Wash and scan as described in Subheading 3.4. 8. Store printed slides in a dry dark place (e.g., in the original plastic slide container) at room temperature until used (see Note 16). 3.3 Microarray Binding Assays (See Note 12)

Perform all incubations with the slide placed on a horizontal surface and protected from light and dust with an opaque cover. Very important, avoid membrane drying between steps. 1. Place a Proplate slide module over the slide and fix the ensemble with two lateral clips (Fig. 1). 2. Add 200 μL of the appropriate blocking buffer for the lectin to be tested (see Subheading 2.3) to completely cover the nitrocellulose pad and incubate for 60 min at room temperature. 3. Remove the blocking buffer by flicking the frame and wash once the wells with washing buffer, using a wash-bottle and flicking to remove the solution. If required, excess fluid may be removed by pipetting. 4. Add a minimum of 100 μL per pad of lectin solution in the selected overlay buffer and incubate for 90 min at room temperature. The use of lectins at 20 μg/mL is recommended for initial testing (see Note 17). For evaluation of carbohydratemediated lectin binding, incubate in parallel a different pad with a minimum of 100 μL of the lectin solution containing the respective hapten, typically at a concentration of 10 mM or above. As blank, also incubate another pad with overlay buffer. 5. Remove overlay solutions by flicking or with the help of a pipette. Wash the pads 4 times as described in step 3. 6. For non-biotinylated lectins, incubate the pads previously overlaid with lectin, lectin + hapten, or buffer with a 1:500 dilution of the indicated biotinylated antibody (see Notes 17 and 18). Then proceed as in step 5. 7. Add to all pads 1 μg/mL of AF647-labeled streptavidin in overlay buffer and incubate at room temperature for 35 min. Incubation with streptavidin should be performed simultaneously in all the pads of a given slide. If the number of previous incubation/washing steps differs among pads, the longest binding assays should be started first (from step 2) and timed to coincide with shorter assays at the final streptavidin incubation step. 8. Remove streptavidin solution by flicking and wash 6 times with washing buffer.

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9. Carefully remove the slide from the holder and rinse thoroughly with Milli-Q water. Let the slide drain and dry in a dark place. 10. Scan for SYTO® 13 and Alexa Fluor 647 fluorescence signals as described in Subheading 3.4. 3.4 Microarray Scanning and Data Analysis

1. For scanning slides after printing, wash the slides by a brief immersion in distilled water to remove any remaining glycerol and let them dry protected from light (see Note 19). 2. Scan the slides using the GenePix Pro 6.1 software and these recommended starting parameters: 100% laser power, 10 μm scan resolution, and photo-multiplier tube (PMT) gain at 280, 250, and 400 for blue (excitation at 488 nm), green (excitation at 532 nm), and red (excitation at 635 nm) lasers, respectively. These parameters should in principle facilitate to get a good signal intensity while avoiding interference with the nitrocellulose intrinsic fluorescence (see Notes 20 and 21). Save the images for later analysis. 3. After the binding assays, scan the slide using the parameters stated in step 2 and save the images. 4. Visually check the quality of the red images obtained. If too low and/or saturated signals are observed, rescan the slide increasing (400–700 range) and/or reducing (300–400 range), respectively, the red laser PMT gain (see Note 22). Save all the images. 5. Open and superimpose the green, blue, and red images obtained before the binding assay and, if necessary, fit the green and red images to the blue one (hereafter referred to as blue reference image). Create 16 blocks in a 2 column
8 row distribution (as in the 16-pad slide), each one containing a grid of 100 μm-diameter spots matching the printing layout (e.g., 12 column
18 row, see Subheading 3.2, step 6). Locate the spots using a fixed circle protocol and check that all the spots have been correctly found. When required, missed spots may be individually located. 6. Quantify and save the file. We recommend subtraction of the local spot background for quantitation. Hide the blocks (see Note 23). 7. For quantitation of post-binding assay images, reopen the blue reference image and fit the images to the reference. Then show the blocks, checking that they are correctly aligned, and quantify as in step 6. Save the file. Repeat as many times as necessary, depending on the number of images obtained in step 4. 8. Use values of median spot intensities minus background for analysis. Normalize SYTO-13 fluorescence intensities (blue

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Fig. 2 Microarray data analysis. Serial dilutions of bacterial samples (1–6) and control (glyco)proteins printed on 16-pad nitrocellulose-coated slides were tested for the binding of a representative lectin in the absence or presence of a specific lectin hapten. A pad incubated with buffer was used as blank. Left panel, median spot intensities of AF647-streptavidin signals (used to monitor lectin binding) for samples printed in duplicate at four dilution levels. Strong and hapten-inhibitable binding to fetuin (F) and asialofetuin (AF), but not to ribonuclease A (RA), evidenced lectin activity. Right panel, correlation of AF647 signals with normalized SYTO-13 fluorescence intensities, which indicate the amount of bacteria actually printed in each spot. Triangles, sample 3; Squares, sample 4; gray symbols, blank. Dose-dependent and hapten-inhibitable binding to certain bacterial samples was observed

signals) based on the labeling yield obtained in Subheading 3.1. Generate tables or graphs correlating red (Alexa-F647) and blue (SYTO-13) values (Fig. 2), using complementary software. 9. For analysis and evaluation of the results: (a) Compare the obtained graphs and tables with source images to identify potential erratic results (due to, e.g., background noise, sample precipitation, partial drying of the membrane, etc.); (b) Check the intensity of the binding to control (glyco)proteins (see Note 5); (c) When significant compared to the values obtained in the lectin binding assay, subtract from the binding signals the values obtained in the corresponding blank pads; and (d) to discriminate carbohydrate and non-carbohydrate-mediated binding, compare the results obtained in the absence and presence of lectin haptens (Fig. 2).

4

Notes 1. Fixing of bacteria (e.g., with 4% paraformaldehyde) is essential to avoid alterations in cell surface components due to changes in the bacterial environment along the analysis. Fixation also

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allows working with pathogenic bacteria in a biosafety level 1 laboratory. 2. Selection of the fluorescent dye used for bacteria labeling should be based on the technical specifications of the microarray scanner and on the dye used for detection of lectin binding (see Subheading 3.3). Thus, SYTO® 13 may be substituted by other DNA/RNA labeling dye, as e.g., SYTO® 82 (orange fluorescent stain). In this case it is necessary to use appropriate filters in the microarray scanner for minimizing interference with the dye used for detection of binding signals. 3. Non-contact printing is important to avoid punching the nitrocellulose membrane with the microarrayer dispensing capillaries. A detailed description of features of the Sprint microarrayer is given in [11]. Alternatively, a manual microarraying system can be used, as described in [11], making the technique more affordable. However, the manual arraying system is not suitable for high-throughput assays. 4. Cy3 is used for monitoring spot location of printed (glyco)proteins [14]. Although the Cy3 NHS ester reacts with protein amino groups, aqueous solutions are readily hydrolyzed to the free acid. Therefore, covalent modification of the glycoproteins is not expected. 5. Fetuin contains a diversity of sialylated N-linked complex-type glycans as well as O-linked glycans that are recognized by many lectins. Ribonuclease B contains N-linked high-mannose-type glycans. Ribonuclease A is not glycosylated. Specific lectin binding to the appropriate control glycoprotein serves as proof of lectin activity. 6. The Proplate module is designed to divide the slide into 16 incubation chambers (Fig. 1), enabling to perform 16 independent binding assays simultaneously. 7. Large collections of biotinylated lectins of known carbohydrate-binding specificities are commercially available (e.g., from Vector Laboratories or EY Laboratories). Suitable buffers and inhibitory haptens for each lectin are indicated by the manufacturers. 8. Many innate immune lectins are commercially available in non-labeled or labeled form (from, e.g., ATGen, Sino Biological Inc., or R&D Systems). Buffers and haptens should be initially selected based on information available in the literature for each particular lectin. Ideally, use conditions previously employed in, e.g., ELISA, FACScan, or histochemical assays. 9. The use of Tween 20 in the blocking and overlay buffers is recommended for ensuring a clean background. If required, it may be substituted by 3% and 1% BSA, respectively.

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10. In case the available primary antibody is not biotinylated, it will be necessary to use a biotinylated secondary antibody, although the specificity of the analysis may decrease due to antibody cross-reactivity. 11. Bacterial labeling with SYTO® 13 is visible to the naked eye, as cells become yellowish. The supernatant should be colorless. If some color is observed, this indicates bacterial lysis, requiring additional washing steps to remove lysis products that could interfere with the results. The maximum of the spectrum of SYTO® 13-stained bacteria upon excitation at 488 nm depends on their DNA/RNA ratio, varying from 507 to 514 nm. To avoid saturation of the fluorescence signal, the use of a narrow slit (1 nm) in the spectrofluorimeter is usually necessary. 12. Wearing a mask is essential for microarray preparation and subsequent binding assays to avoid contamination due to micro-spitting. 13. For accurate printing, samples need to be prepared in a highdensity buffer. Although this may complicate sample preparation, it is an advantage when printing bacterial suspensions as it reduces bacteria sedimentation, thus ensuring sample homogeneity during printing. In addition, the presence of 0.06% Triton X-100 reduces sample aggregation and ensures the cleanliness of the printing system. 14. Centrifugation of sample plates is important to avoid the presence of bubbles, which would cause problems in sample aspiration, and undesired particles that could block the microarrayer capillaries. To prevent sedimentation of bacteria, a short spinning at 200
g is suggested. 15. Before arraying, run a routine microarrayer cleaning and print a test slide with buffer to check a correct performance. 16. Slides can be stored over a period of 1 year or more with no discernible loss of performance. 17. The concentration of biotin-labeled lectins and antibodies used for the binding assays may need to be adjusted depending on the results of initial tests. 18. If the primary antibody is not biotinylated, incubate next for 60 min with a 1:1000 dilution of the biotinylated secondary antibody, and then proceed as in step 5. Alternatively, a pre-complexed mixture of the two antibodies can be used in a single incubation step. 19. Scanning in the presence of glycerol results in a progressive loss of SYTO® 13 fluorescence intensity. In addition, glycerol quenches the intrinsic fluorescence of the nitrocellulose membrane, producing a black circle around printed bacteria that complicates spot location with the scanner software. Washing

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for glycerol removal results in a decrease of Cy3 signal intensities, but the remaining Cy3 fluorescence is sufficient for spot tracing. Although once glycerol is removed there is no significant loss of SYTO® 13 signal intensity, we still recommend to scan first with the blue laser and next with the green and red lasers. 20. The gain of the photo-multiplier tube has to be adjusted in order to obtain the best signal intensities while avoiding interference of the nitrocellulose intrinsic fluorescence and spot signal saturation (white spots). 21. Red scan before the binding assay is not necessary but recommended to check possible interference of the intrinsic fluorescence of the samples. 22. The signals in the different pads of a given slide may substantially differ in intensity. Therefore, it may be necessary to rescan the slide using distinct pad-customized PMT gains. 23. Hiding the blocks prevents displacements of the grid that may occur when fitting the post-binding assay images to the reference image using the GenePix Pro 6.1 software.

Acknowledgments We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grant BFU201570052-R), the Ministry of Science, Innovation, and Universities (RTI2018-099985-B-I00), and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). References 1. Salton MRJ, Kim KS (1996) Structure. In: Baron S (ed) Medical microbiology. Galveston, TX 2. Kilcoyne M, Twomey ME, Gerlach JQ, Kane M, Moran AP, Joshi L (2014) Campylobacter jejuni strain discrimination and temperature-dependent glycome expression profiling by lectin microarray. Carbohydr Res 389:123–133. https://doi.org/10.1016/j. carres.2014.02.005 3. Casals C, Campanero-Rhodes MA, Garcı´aFojeda B, Solı´s D (2018) The role of collectins and galectins in lung innate immune defense. Front Immunol 9:1998. https://doi.org/10. 3389/fimmu.2018.01998 4. Sukhithasri V, Nisha N, Biswas L, Kumar VA, Biswas R (2013) Innate immune recognition of

microbial cell wall components and microbial strategies to evade such recognitions. Microbiol Res 168(7):396–406. https://doi.org/ 10.1016/j.micres.2013.02.005 5. Wesener DA, Dugan A, Kiessling LL (2017) Recognition of microbial glycans by soluble human lectins. Curr Opin Struct Biol 44: 168–178. https://doi.org/10.1016/j.sbi. 2017.04.002 6. Zheng RB, Je´gouzo SAF, Joe M, Bai Y, Tran HA, Shen K, Saupe J, Xia L, Ahmed MF, Liu YH, Patil PS, Tripathi A, Hung SC, Taylor ME, Lowary TL, Drickamer K (2017) Insights into interactions of mycobacteria with the host innate immune system from a novel array of synthetic mycobacterial glycans. ACS Chem

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Biol 12(12):2990–3002. https://doi.org/10. 1021/acschembio.7b00797 7. Stowell SR, Arthur CM, McBride R, Berger O, Razi N, Heimburg-Molinaro J, Rodrigues LC, Gourdine J-P, Noll AJ, von Gunten S, Smith DF, Knirel YA, Paulson JC, Cummings RD (2014) Microbial glycan microarrays define key features of hostmicrobial interactions. Nat Chem Biol 10(6):470–476 8. Wesener DA, Wangkanont K, McBride R, Song X, Kraft MB, Hodges HL, Zarling LC, Splain RA, Smith DF, Cummings RD, Paulson JC, Forest KT, Kiessling LL (2015) Recognition of microbial glycans by human intelectin-1. Nat Struct Mol Biol 22(8): 603–610 9. Palma AS, Feizi T, Zhang Y, Stoll MS, Lawson AM, Dı´az-Rodrı´guez E, Campanero-Rhodes MA, Costa J, Gordon S, Brown GD, Chai W (2006) Ligands for the beta-glucan receptor, Dectin-1, assigned using “designer” microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides. J Biol Chem 281(9):5771–5779 10. Campanero-Rhodes MA, Llobet E, Bengoechea JA, Solı´s D (2015) Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors. RSC Adv 5(10):7173–7181. https://doi. org/10.1039/c4ra14570d

11. Kalograiaki I, Campanero-Rhodes MA, Proverbio D, Euba B, Garmendia J, Aastrup T, Solı´s D (2018) Bacterial surface glycans: microarray and QCM strategies for glycophenotyping and exploration of recognition by host receptors. Methods Enzymol 598: 37–70 12. Kalograiaki I, Euba B, Proverbio D, Campanero-Rhodes MA, Aastrup T, Garmendia J, Solı´s D (2016) Combined bacteria microarray and quartz crystal microbalance approach for exploring glycosignatures of nontypeable Haemophilus influenzae and recognition by host lectins. Anal Chem 88(11): 5950–5957 13. Kalograiaki I, Euba B, Ferna´ndez-Alonso MDC, Proverbio D, St Geme JW 3rd, ˜ ada FJ, Solı´s D Aastrup T, Garmendia J, Can (2018) Differential recognition of Haemophilus influenzae whole bacterial cells and isolated lipooligosaccharides by galactose-specific lectins. Sci Rep 8(1):16292 14. Campanero-Rhodes MA, Childs RA, Kiso M, Komba S, Le Narvor C, Warren J, Otto D, Crocker PR, Feizi T (2006) Carbohydrate microarrays reveal sulphation as a modulator of siglec binding. Biochem Biophys Res Commun 344(4):1141–1146

Chapter 10 Tissue Glycome Mapping: Lectin Microarray-Based Differential Glycomic Analysis of Formalin-Fixed Paraffin-Embedded Tissue Sections Chiaki Nagai-Okatani, Xia Zou, Atsushi Matsuda, Yoko Itakura, Masashi Toyoda, Yan Zhang, and Atsushi Kuno Abstract Lectin microarray (LMA) is a high-sensitive glycan analysis technology used to obtain global glycomic profiles of both N- and O-glycans attached not only to purified glycoproteins but also to crude glycoprotein samples. Through additional use of laser microdissection (LMD) for tissue collection, we developed an LMA-based glycomic profiling technique for a specific type of cells in a tiny area of formalin-fixed paraffinembedded (FFPE) tissue sections. This LMD–LMA method makes it possible to obtain reproducible tissue glycomic profiles that can be compared with each other, using a unified protocol for all procedures, including FFPE tissue preparation, tissue staining, protein extraction and labeling, and LMA analysis. Here, we describe the standardized LMD–LMA procedure for a “tissue glycome mapping” approach, which facilitates an in-depth understanding of region- and tissue-specific protein glycosylation. We also describe potential applications of the spatial tissue glycomic profiles, including histochemical analysis for evaluating distribution of lectin ligands and a fluorescence LMD–LMA method for cell type-selective glycomic profiling using a cell type-specific probe, composed of a lectin and an antibody. The protocols presented here will accelerate the effective utilization of FFPE tissue specimens by providing tissue glycome maps for the discovery of the biological roles and disease-related alterations of protein glycosylation. Key words Tissue glycome mapping, Formalin-fixed paraffin-embedded tissue section, Laser microdissection, Lectin microarray, Histochemistry, Cell type-selective analysis

1

Introduction As one of the most common post-translational modifications, protein glycosylation plays pivotal functional roles in various biological events [1]. Since this modification highly increases the variation and complexity in their structures, analysis of the variety and density of glycans attached to proteins is still challenging. Accordingly, development of simple and rapid methods for analyzing glycoproteins based on several technologies with different concepts is of paramount importance for understanding their biological and

Michelle Kilcoyne and Jared Q. Gerlach (eds.), Glycan Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 2460, https://doi.org/10.1007/978-1-0716-2148-6_10, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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pathological functions [2, 3]. To meet this demand, lectin microarray (LMA) has been developed, which was initially introduced in 2005 [4, 5]. LMA is a high-sensitive glycan analysis technology used to obtain global glycomic profiles of glycans attached on not only purified glycoproteins but also crude glycoprotein samples [3, 6]. Compared to mass spectrometry-based glycomic analysis, LMA-based analysis has an advantage in that both N- and Oglycoproteins can be simultaneously assessed in a simple and rapid procedure. Conversely, as LMA-based glycomic profiles are presented as the signal patterns of multi-lectin–glycoprotein interactions, LMA provides less detailed information on glycan structures. Thus, these two types of analytical methods are considered to have complementary roles in glycomic analysis [2, 3]. As a common type of tissue specimens, formalin-fixed paraffinembedded (FFPE) tissues have an advantage of a long storage time compared to frozen tissues, and thus are a highly valuable source for morphological observations and molecular analyses in biological and pathological studies. To effectively utilize FFPE tissue sections, where glycans and their carrier proteins are well preserved, for obtaining LMA-based glycomic profiles related to clinical and pathological characteristics, an all-in-one procedure was developed in 2008 [7], in which tissue collection was performed manually. The utility of this method for the detection of disease-related alterations in protein glycosylations has been firmly demonstrated in several studies [8–11]. To improve the reliability and usefulness of the LMA-based tissue glycomic analysis, since 2012, efforts has been made towards developing a standardized procedure in combination with laser microdissection (LMD), which allows an accurate and precise collection of tissue fragments from a tiny area by direct visualization [12]. The feasibility and utility of this standardized LMD–LMA method has been demonstrated using FFPE sections of five organs (the brain, liver, kidney, spleen, and testis) of normal mice, where the differential glycomic profiling of intra- and intertissues showed region-specific patterns of protein glycosylation on multiple tissue sections [13]. Using the standardized method, we revealed characteristics in glycan structures that are specifically expressed in fibrotic cardiac tissues of dilated cardiomyopathy model mice [14]. In these analyses using mouse tissue sections, the collection of each 0.5–1.0 mm2 area of 5 μm-thick tissue fragments was enough for differential glycomic profiling. Taking advantage of the high sensitivity of the standardized LMD–LMA method, we employed this method for a “tissue glycome mapping” approach, which aims to visualize the glycomic profiles of each area of the tissue sections [3]. As a freely available atlas obtained by this approach, the tissue glycomic profile data of nine tissues (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of normal mice was recently provided using a newly

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developed online tool, called “LM-GlycomeAtlas” (https://glycosmos.org/lm_glycomeatlas/index) [15, 16]. Here, we provide a protocol of the standardized LMD–LMA method for tissue glycome mapping using FFPE tissue sections. The protocol consists of four main steps: (1) preparation and hematoxylin staining of FFPE tissue sections, (2) LMD-assisted tissue dissection based on morphological observation, (3) extraction and fluorescent labeling of protein samples, and (4) LMA analysis and data processing. The most important requirement for effective tissue glycome mapping is to obtain reliable glycomic profiles from multiple regions and tissues that can be compared with each other. Because proteins stored in FFPE tissues are highly cross-linked, the conditions of tissue fixation and antigen retrieval treatment may influence the degree of protein extraction [17]. In addition, tertiary structures of proteins obtained from FFPE tissue sections may vary depending on these conditions [17], which results in the differences in accessible glycans and functional groups for labeling. In this situation, it is essential to perform all the steps in a unified protocol for obtaining comparable tissue glycome mapping data. Accordingly, providing the detailed protocol here will accelerate the effective utilization of FFPE tissue specimens for LMA-based glycomic analyses of glycoproteins. The resulting tissue glycome mapping data can be utilized for selecting a lectin that recognizes glycans expressing in a specific type of cells and site of interest. The cell type- and site-specificity of the lectin can be evaluated by lectin histochemistry using FFPE tissue sections, which also provides information regarding where lectin ligands distribute on the cell surface. After the histochemical validation, the cell type-specific lectin can be utilized as a probe for visualization of the cells of interest. As an advanced LMD–LMA method for cell type-selective tissue glycomic profiling, we recently developed a procedure for tissue collection by LMD under fluorescent histochemical visualization using a cell type-specific probe such as a lectin and an antibody against molecular markers [18]. Accordingly, we also provide here the optional protocols of these experiments related to tissue glycome mapping, including lectin histochemical analysis and cell type-selective glycomic profiling by the fluorescence LMD–LMA method. Incidentally, a lectin selected by tissue glycome mapping can also be used as an affinity probe to collect glycoproteins carrying the lectin-binding glycans. For the further glycan analysis of specific glycoproteins, an antibody-assisted glycan profiling method can be employed [19, 20], for which a detailed protocol has been provided previously [21].

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Materials Prepare all solutions using ultrapure water and analytical grade reagents. Prepare and store all reagents and materials at room temperature unless indicated otherwise. Follow all safety and waste disposal regulations.

2.1 Preparation of FFPE Tissue Sections for Tissue Dissection

1. FFPE tissue blocks (see Note 1). 2. Glass slides; polyethylene naphthalate (PEN)- and polyphenylene sulfide (PPS)-membrane glass slides (Leica Microsystems, Wetzlar, Germany) for preparation of tissue sections for staining with hematoxylin and fluorescence histochemical probes, respectively. 3. 0.1% (w/v) poly-L-lysine solution (Sigma-Aldrich, St. Louis, MO, USA). 4. Sliding microtome (SM2000R; Leica Microsystems) equipped with disposable blades (S35; Feather Safety Razor, Osaka, Japan).

2.2 Deparaffinization and Hematoxylin Staining of FFPE Tissue Sections

1. Solvents for deparaffinization and rehydration: xylene and 100%, 90%, 70%, and 50% (v/v) ethanol. 2. Mayer’s hematoxylin solution (Fujifilm Wako Pure Chemical, Osaka, Japan). 3. Fluorescence microscope (BZ-X710; Keyence, Osaka, Japan), which is used for bright-field observation.

2.3 LMD-Assisted Tissue Section

1. LMD system (LMD7000) attached (DM6000B) (see Note 2).

to

a

microscope

2. Tissue collection tube (0.5-mL volume; Axygen, Union City, CA, USA). 2.4 Tissue Protein Extraction for LMA Analysis

1. 10 mM citrate buffer (pH 6.0): Dissolve 2.1 g of citric acid monohydrate in 100 mL water to obtain 0.1 M citric acid solution. Dissolve 14.7 g of trisodium citrate dihydrate to obtain 0.1 M sodium citrate solution. Mix 3.6 μL of 0.1 M citric acid solution, 16.4 μL of 0.1 M sodium citrate solution, and 180 μL water to obtain 200 μL of the working buffer, which is used for one sample (see Note 3). Check that the pH of the resulting solution is 6.0  0.2. Use within 1 day. 2. Dulbecco’s PBS (D-PBS): Dissolve 0.2 g NaH2PO4, 1.15 g Na2HPO4, 0.2 g KCl, and 8 g NaCl in approximately 900 mL of water, and then make up to 1 L with water. Check that the pH of the resulting solution is 7.4  0.2. Store at 4  C. 3. 50% (w/v) slurry of microcrystalline cellulose, a co-precipitant: Weigh 50 μg of microcrystalline cellulose (Avicel PH-101;

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Sigma-Aldrich) in a 1.5-mL tube and suspend in 1 mL D-PBS (see Note 4). After mixing with a vortex mixer, centrifugate at 20,000  g for 1 min at 4  C and discard the supernatant. Repeat this wash step more twice. Suspend the resultant pellet with 50 μL D-PBS to obtain the working slurry. Use within 1 day. 4. D-PBS containing 1% (v/v) Nonidet P-40 (NP-40): Mix 5 μL NP-40 and 495 μL D-PBS to obtain 500 μL of the working solution (see Note 5). Use within 1 day. 5. Ultrasonic bath sonicator (Tamagawa Seiki, Nagano, Japan) (see Note 6). 6. Low-retention tips (epT.I.P.S.; Eppendorf, Hamburg, Germany) and tubes (Protein LoBind; Eppendorf) (see Note 7). 2.5 Fluorescence Labeling and LMA Analysis of Tissue Protein Samples

1. Autopipettes (Finnpipette Novus electronic single-channel pipettes, 1–10 μL and 100–1000 μL; Thermo Fisher Scientific, Waltham, MA, USA). 2. A working Cy3-succinimidyl ester (Cy3-SE) dye (10 μg protein equivalent of Cy3-SE/tube): Dissolve 1 mg protein equivalent of Cy3-SE (GE Healthcare, Buckinghamshire, UK) in N,Ndimethylformamide that is well dehydrated using molecular sieves (4A 1/16; Nacalai Tesque, Kyoto, Japan) before use. Dispense an aliquot of the resulting dye solution into an eightstrip 0.2-mL tube (5 μL/tube) using the 1–10 μL autopipette and dry up using a centrifugal evaporator. Store the working dye with a dehydrating agent at 4  C in the dark. 3. Probing buffer: 25 mM Tris-HCl, pH 7.5, containing 137 mM NaCl, 2.7 mM KCl, 500 mM glycine, 1 mM CaCl2, 1 mM MnCl2, and 1% Triton X-100. Store at 4  C. 4. Lectin array chips (LecChip™ Ver.1.0; GlycoTechnica, Yokohama, Japan), which consist of triplicate spots of 45 lectins (Table 1). 5. Humidified shading chamber (dark orange; Cosmo Bio, Tokyo, Japan). 6. Low-retention tips and tubes (see Subheading 2.4, item 6). 7. Evanescent-field fluorescence scanner (GlycoStation™ Reader 1200; GlycoTechnica) [6]. 8. LMA data analysis software (GlycoStation™ Tools Pro Suite 1.5; GlycoTechnica).

2.6 Deparaffinization and Fluorescence Histochemical Staining of FFPE Tissue Sections

1. Solvents for deparaffinization and rehydration (see Subheading 2.2, item 1). 2. Antigen retrieval buffer: Target Retrieval Solution, Citrate (pH 6.0) (Agilent Technologies, Santa Clara, CA, USA) (see Note 8).

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Table 1 Abbreviations and carbohydrate specificities of 45 lectins on the LecChip Ver.1.0 No. Lectin

Origin

Binding specificitya

1

LTL

Lotus tetragonolobus

Fucα1–3GlcNAc, Sia-Lex and Lex

2

PSA

Pisum sativum

Fucα1-6GlcNAc and α-Man

3

LCA

Lens culinaris

Fucα1-6GlcNAc, α-Man and α-Glc

4

UEA-I

Ulex europaeus

Fucα1–2LacNAc

5

AOL

Aspergillus oryzae

Terminal α-Fuc, Sia-Lex and Lex

6

AAL

Aleuria aurantia

Terminal α-Fuc, Sia-Lex and Lex

7

MAL-I

Maackia amurensis

Siaα2-3Gal

8

SNA

Sambucus nigra

Siaα2-6Gal/GalNAc

9

SSA

Sambucus sieboldiana

Siaα2-6Gal/GalNAc

10 TJA-I

Trichosanthes japonica

Siaα2-3Galβ1-4GlcNAcβ-R

11 PHA-L

Phaseolus vulgaris

Tri- and tetra-antennary complex-type N-glycan

12 ECA

Erythrina cristagalli

Lac/LacNAc

13 RCA120

Ricinus communis

Lac/LacNAc

14 PHA-E

Phaseolus vulgaris

NA2 and bisecting GlcNAc

15 DSA

Datura stramonium

(GlcNAc)n, polyLacNAc and LacNAc (NA3, NA4)

16 GSL-II

Griffonia simplicifolia

Agalactosylated N-glycan

17 NPA

Narcissus pseudonarcissus

Non-substituted α1-6Man

18 ConA

Canavalia ensiformis

α-Man (inhibited by presence of bisecting GlcNAc)

19 GNA

Galanthus nivalis

Non-substituted α1-6Man

20 HHL

Hippeastrum hybrid

Non-substituted α1-6Man

21 ACG

Agrocybe cylindracea

Siaα2-3Galβ1-4GlcNAc

22 TxLC-I

Tulipa gesneriana

Man3, bi- and tri-antennary complex-type N-glycan, GalNAc

23 BPL

Bauhinia purpurea alba

Galβ1–3GalNAc and NA3, NA4

24 TJA-II

Trichosanthes japonica

Fucα1–2Gal, β-GalNAc > NA3, NA4

25 EEL

Euonymus europaeus

Galα1–3[Fucα1–2]Gal > Galα1–3Gal

26 ABA

Agaricus bisporus

Galβ1–3GalNAcα-Thr/Ser (T) and sialyl-T

27 LEL

Lycopersicon esculentum

(GlcNAc)n and polyLacNAc

28 STL

Solanum tuberosum

(GlcNAc)n and polyLacNAc

29 UDA

Urtica dioica

(GlcNAc)n and polyLacNAc

30 PWM

Phytolacca americana

(GlcNAc)n and polyLacNAc

31 Jacalin

Artocarpus integrifolia

Galβ1–3GalNAcα-Thr/Ser (T) and GalNAcα-Thr/Ser (Tn)

32 PNA

Arachis hypogaea

Galβ1–3GalNAcα-Thr/Ser (T) (continued)

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Table 1 (continued) No. Lectin

Origin

Binding specificitya

33 WFA

Wisteria floribunda

Terminal GalNAc (e.g., GalNAc β1-4GlcNAc)

34 ACA

Amaranthus caudatus

Galβ1–3GalNAcα-Thr/Ser (T)

35 MPA

Maclura pomifera

Galβ1–3GalNAcα-Thr/Ser (T) and GalNAcα-Thr/Ser (Tn)

36 HPA

Helix pomatia

Terminal GalNAc

37 VVA

Vicia villosa

α-, β-linked terminal GalNAc and GalNAcα-Thr/Ser (Tn)

38 DBA

Dolichos biflorus

GalNAcα-Thr/Ser (Tn) and GalNAcα1–3GalNAc

39 SBA

Glycine max

Terminal GalNAc (especially GalNAcα1–3Gal)

40 Calsepa

Calystega sepiem

Man and Maltose

41 PTL-I

Psophocarpus tetragonolobus

α-GalNAc and Gal

42 MAH

Maackia amurensis

Siaα2-3Galβ1–3[Siaα2-6GalNAc]α-R

43 WGA

Triticum vulgaris

(GlcNAc)n and multivalent Sia

44 GSL-IA4

Griffonia simplicifolia

α-GalNAc and GalNAcα-Thr/Ser (Tn)

45 GSL-IB4

Griffonia simplicifolia

α-Gal

a

Binding specificities are based on Lectin Frontier Database (LfDB; https://acgg.asia/lfdb2)

3. 10 mM phosphate buffered saline (PBS): Dissolve 0.35 g NaH2PO4, 1.28 g Na2HPO4, and 8 g NaCl in approximately 900 mL of water, and then make up to 1 L with water. Check that the pH of the resulting solution is 7.4  0.2. 4. Streptavidin–biotin blocking kit (Vector Laboratories, Burlingame, CA, USA), which contains streptavidin and biotin solutions. 5. Carbo-free blocking solution (Vector Laboratories). The working solution is obtained by diluting the concentrated stock solution tenfold with water. 6. Biotinylated lectins and antibodies: Apply 50–200 μg of an unlabeled protein to a labeling reaction using the biotinlabeling kit-NH2 (Dojindo, Kumamoto, Japan) (see Note 9). Store at 4  C. 7. Fluorescein isothiocyanate (FITC)-labeled lectin of interest, which is used for fluorescence double-staining analysis (see Subheading 3.2).

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8. Alexa Fluor 647-conjugated streptavidin (Thermo Fisher Scientific), which is used for fluorescence double-staining using a lectin and an antibody. 9. Alexa Fluor 594-conjugated streptavidin (Thermo Fisher Scientific), which is used for fluorescence LMD–LMA method (see Note 10). 10. ProLong Gold antifade mountant (Thermo Fisher Scientific). 11. Fluorescence microscope (see Subheading 2.2, item 3).

3

Methods Carry out all procedures at room temperature (approximately 25  C) unless otherwise specified.

3.1 LMD–LMA Analysis of Hematoxylin-Stained Tissue Sections 3.1.1 Preparation of Tissue Sections

1. Spread 80 μL of 0.1% poly-L-lysine solution on a PEN-membrane glass slide and air-dry for 0.5–1 day (see Note 11). 2. Slice an FFPE tissue block using the sliding microtome. Float the resulting 5 μm-thick sections on the surface of pre-heated water at 42  C in a water bath so that the sections flatten out and then mount on the poly-L-lysine-coated slide. 3. Dry the slide overnight on a hot plate at 42  C.

3.1.2 Hematoxylin Staining

1. Deparaffinize and rehydrate the slide by immersion in the following solutions in the following order with the specified times: xylenes, three times at 5 min each; 100% ethanol, three times at 5 min each; 90% ethanol, one time at 3 min; 70% ethanol, one time at 3 min; 50% ethanol, one time at 3 min; water, twice at 1 min each. 2. Immerse the slide in Mayer’s hematoxylin solution for 1–10 min and wash with tap water for 10 min (see Note 12). 3. Dry the slide using a hot air dryer (see Note 13). Store in the dark until use (see Note 14).

3.1.3 LMD-Assisted Tissue Dissection

1. Before tissue dissection, capture a high-resolution image of the whole section under bright-field optics using the BZ-710 microscopy, which will be used for determination of dissected areas (see Note 15). 2. Attach the hematoxylin-stained section and tissue collection tubes to the holders of the LMD system (see Note 16). 3. Dissect tissue fragments (total area of 0.5–1.0 mm2 per tube) under bright-field optics (see Note 17), using the following conditions: magnification, 6.3–20; laser power, 15–30;

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Fig. 1 Example images of LMD-assisted tissue dissection of hematoxylin-stained sections. (a) Bright-field image of a whole pancreatic section of 8-week-old C57BL/6J male mouse after dissection. (b) Tissue dissection images of the areas indicated in (a) and images of the corresponding areas of the serial section stained with hematoxylin and eosin (HE). The images of the other areas on the hematoxylin-stained pancreatic section are available in the LM-GlycomeAtlas (https://glycosmos.org/lm_glycomeatlas/index). Scale bars, 300 μm

aperture, 6; speed, 1 (see Note 18). Collect the tissue fragments into the caps of collection tubes. 4. Close the cap gently in the state of the cap being located at the bottom. 5. After all the dissection procedures, re-capture a high-resolution image of the whole section as described above, which will be used for annotation of the dissected areas. 6. Figure 1 shows example images of tissue dissection from hematoxylin-stained sections. 3.1.4 Protein Extraction and Fluorescence Labeling

To ensure uniform conditions and efficiency in protein extraction and labeling, which may influence the resulting glycomic profiles, these steps should be performed as simultaneously as possible for all the samples to be compared. Perform the same manipulation for one tube without tissue samples as a negative control, for evaluating potential contamination derived from microcrystalline cellulose and buffers. 1. Spin down tissue fragments at the bottom of the tube by centrifugation at 20,000  g for 1 min at 4  C (see Note 19). 2. Perform heat-denaturing of the tissue fragments as follows. First, add 200 μL of 10 mM citrate buffer (pH 6.0) and

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centrifuge at 20,000  g for 1 min at 4  C. Thereafter, incubate the buffer-containing tube on a dry heat block at 95  C for 1 h for heat-denaturation of tissue fragments and centrifuge at 20,000  g for 1 min at 4  C. 3. Add 4 μL of 50% slurry of microcrystalline cellulose, mix well with a vortex mixer, and then centrifuge at 20,000  g for 1 min at 4  C. 4. Carefully aspirate 190 μL of the supernatant (see Note 20), add 190 μL D-PBS, mix well with a vortex mixer, and centrifuge at 20,000  g for 1 min at 4  C, for washing of the tissue fragments. 5. Carefully aspirate 190 μL of the supernatant, add 10 μL of D-PBS containing 1% NP-40 (see Note 20), mix well with a vortex mixer, and chill the tube on ice for 1 min. 6. Hold the tube by hand. Sonicate the tube by the ultrasonic bath sonicator for 3 s at 20  C (see Note 21), and chill the tube on ice for 1 min. Repeat these procedures twice more (see Note 22). After the sonication, incubate the tube for 1 h on ice. 7. After the protein extraction step, centrifuge the tube at 20,000  g for 1 min at 4  C and collect 20 μL of the supernatant in a low-retention tube (see Note 23). Make up to 20 μL with D-PBS. If necessary, store the protein extract at 30  C until use (see Note 24). 8. Dissolve the working Cy3-SE dye (10 μg protein equivalent) with 20 μL of the protein extract by pipetting (see Note 25) and incubate the reaction solution in a low-retention tube for 1 h in the dark. 9. After the labeling reaction, add 80 μL of the probing buffer and incubate for 2 h in the dark, to quench the reaction. Keep the Cy3-labeled protein sample on ice for subsequent analysis within 1 day, or store at –30  C until use (see Note 24). 3.1.5 LMA Analysis of Cy3-Labeled Protein Samples

1. Take out a lectin array chip (see Note 26) from a storage bag after it reaches room temperature. Wash the chip three times with 60 μL/well of the probing buffer (see Note 27). Add 60 μL/well of the probing buffer and keep the chip in the humidified shading chamber until use (see Note 28). 2. Dilute the Cy3-labeled protein samples (0.5–1.0 mm2 tissue fragment equivalent in 100 μL) with the probing buffer, if necessary (see Note 29). 3. Entirely discard the solution in the chip and immediately apply the samples to each well (60 μL/well). 4. Place the chip in the humidified shading chamber and incubate overnight at 20  C with continuous shaking (60 rpm) using a reciprocating shaker.

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Fig. 2 The array panel (a) and an example scan image with the grids of the LecChip Ver.1.0 (b). The abbreviations of the lectins are indicated in Table 1. BG, background. Note that the grids (small yellow circles) should be adjusted to appropriately locate (see Note 30)

5. Discard the sample solution and wash the chip three times with 60 μL/well of the probing buffer. 6. After adding 60 μL/well of the probing buffer, scan the chip using the evanescent-field fluorescence scanner in the following conditions: exposure time, 199 ms; gain, stepwise acquirement of every 10 between 55 and 125. This serial-scanning results in obtaining eight scan images for one chip. 7. Figure 2 shows an example scanning image of the lectin array chip. 3.1.6 LMA Data Processing

1. Calculate the net intensity of each spot on the lectin array chip using the LMA data analysis software (see Note 30), where a grid size is set to 12. In this analysis, the same grid is applied to the eight scan images of one chip. 2. Export text-format report files containing the net intensity values obtained with each gain for each sample. 3. Combine the net intensity data obtained from the scan images of the eight gain conditions for all the samples of one chip into one Excel-format file. 4. Select an optimal gain condition for each sample to obtain glycomic profiles so that the net intensities are