Methods in molecular biology vol 1588 protein carbohydrate interactions methods and protocols

Methods in Molecular Biology 1588 D Wade Abbott Alicia Lammerts van Bueren Editors ProteinCarbohydrate Interactions Methods and Protocols Methods in Molecular Biology Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hateld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Protein-Carbohydrate Interactions Methods and Protocols Edited by D Wade Abbott Agriculture and Agri-Food Canada, Lethbridge, AB, Canada Alicia LammertsvanBueren Biotechnology Institute, University of Groningen, Groningen, The Netherlands Editors D Wade Abbott Agriculture and Agri-Food Canada Lethbridge, AB, Canada Alicia LammertsvanBueren Biotechnology Institute University of Groningen Groningen, The Netherlands ISSN 1064-3745 ISSN 1940-6029(electronic) Methods in Molecular Biology ISBN 978-1-4939-6898-5ISBN 978-1-4939-6899-2(eBook) DOI 10.1007/978-1-4939-6899-2 Library of Congress Control Number: 2017933839 â Springer Science+Business Media LLC 2017 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, express 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 Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, NewYork, NY 10013, U.S.A Preface Protein-carbohydrate interactions are involved in diverse processes required for life, including the microbial degradation of plant biomass and marine polysaccharides and human health and nutrition Understanding and predicting how carbohydrates are recognized and modified by carbohydrate-active enzymes (i.e., CAZymes) therefore is an important area of basic research that spans multiple disciplines and holds vast promise for informing future innovations in renewable resource utilization and medicine Since the turn of the millennia, the field of protein-carbohydrate interactions has been transformed by high-throughput and ultrasensitive instrumentation, which has enabled us to study complex carbohydrate utilization systems at the levels of metagenomes, metatranscriptomes, and metaproteomes This increase in technology has opened new doors for CAZyme discovery and application Here within we will provide a wide-ranging resource for studying protein-carbohydrate interactions that extends from traditional biochemical methods to state-of-the-art techniques, both of which will continue to propel the field forward in the coming years In particular, this volume will focus on four different research themes Part I describes methods for screening and quantifying CAZyme activity These chapters will survey each class of CAZyme, including glycoside hydrolases (Chap 1, Copper-ư Bicinchoninic Acid; Chap 2, High-Performance Anion-Exchange Chromatography; and Chap 3, 3,5-Dinitrosalicylic Acid Assays), polysaccharide lyases (Chap 4), carbohydrate esterases (Chap 5), glycosyltransferases (Chap 6), and lytic polysaccharide monooxygenases (Chap 7) In addition, a method for investigating carbohydrate depolymerization by cellulosomes, which can contain multiple enzyme classes and activities (Chap 8), is provided Part II contains methods for investigating the interactions between proteins and carbohydrate ligands These techniques include affinity gel electrophoresis of catalytic modules (Chap 9), microscale thermophoresis (Chap 10), and NMR spectroscopy (Chap 11) The final chapter in this section describes current methods for detecting the biomechanical activity of expansions (Chap 12), a class of proteins involved in the loosening of plant cell wall networks Part III discusses methods for the visualization of carbohydrates and protein-ư carbohydrate complexes These chapters include a novel bioinspired plant cell wall assembly for measuring protein interactions by fluorescence (Chap 13), using carbohydrate-binding modules as probes within plant cell walls (Chap 14), and investigating the subcellular localization of CAZymes within Gram-negative bacteria (Chap 15) These are followed by three different methods for investigating carbohydrate structure First is a method for using Fourier transform mid-infrared spectroscopy to characterize the composition of plant cell walls (Chap 16); this is followed by methods for studying fluorescent glycans by electrophoresis (Chap 17) and capillary electrophoresis (Chap 18) Finally, Part IV focuses on structural and omics approaches for studying systems of CAZymes First, a dissect and build approach for determining multimodular CAZyme structure involving combinatorial small-angle X-ray scattering and X-ray crystallography is described (Chap 19) followed by methods describing the development of omics tech- v vi Preface niques to identifying novel CAZyme systems using metagenomics (Chap 20), transcriptomics (Chap 21), and proteomics (Chapter 22) approaches We anticipate that this collection of methods for studying carbohydrate modification and protein-carbohydrate interactions will be a valuable resource to the glycomics research community As the field continues to advance, methods included within this volume will have utility for illuminating the biology of glycomics, driving biotechnological innovation, and developing solutions for human health and for sustainable resources within the emerging green economy Lethbridge, AB, Canada Groningen, The Netherlands D.WadeAbbott AliciaLammertsvanBueren Contents Preface v Contributors ix Part IAnalysis of Carbohydrate-Active Enzyme Activity A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay forGlycoside Hydrolases Gregory Arnal, Mohamed A Attia, Jathavan Asohan, and Harry Brumer Quantitative Kinetic Characterization ofGlycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC) Nicholas McGregor, Gregory Arnal, and Harry Brumer Measuring Enzyme Kinetics ofGlycoside Hydrolases Using the3,5-Dinitrosalicylic Acid Assay Lauren S McKee An Improved Kinetic Assay fortheCharacterization ofMetal-Dependent Pectate Lyases Darryl R Jones, Richard McLean, and D WadeAbbott Colorimetric Detection ofAcetyl Xylan Esterase Activities Galina Mai-Gisondi and Emma R Master Methods forDetermining Glycosyltransferase Kinetics Maria Ngo and Michael D.L Suits Analyzing Activities ofLytic Polysaccharide Monooxygenases by Liquid Chromatography andMass Spectrometry Bjứrge Westereng, Magnus ỉ Arntzen, Jane Wittrup Agger, Gustav Vaaje-Kolstad, and Vincent G.H Eijsink Carbohydrate Depolymerization by Intricate Cellulosomal Systems Johanna Stern, Lior Artzi, Sarah Moraùs, Carlos M.G.A Fontes, and Edward A Bayer 15 27 37 45 59 71 93 Part IIAnalysis of Protein-Carbohydrate Interactions Affinity Electrophoresis forAnalysis ofCatalytic Module-ưCarbohydrate Interactions 119 Darrell Cockburn, Casper Wilkens, and Birte Svensson 10 Quantifying CBM Carbohydrate Interactions Using Microscale Thermophoresis 129 Haiyang Wu, Cộdric Y Montanier, and Claire Dumon 11 Characterization ofProtein-Carbohydrate Interactions byNMR Spectroscopy 143 Julie M Grondin, David N Langelaan, and Steven P Smith vii viii Contents 12 Measuring theBiomechanical Loosening Action ofBacterial Expansins onPaper andPlant Cell Walls 157 Daniel J Cosgrove, Nathan K Hepler, Edward R Wagner, and Daniel M Durachko Part IIIVisualization of Carbohydrates and Protein-Carbohydrate Complexes 13 Bioinspired Assemblies ofPlant Cell Walls forMeasuring Protein-Carbohydrate Interactions by FRAP Gabriel Paởs 14 CBMs asProbes toExplore Plant Cell Wall Heterogeneity Using Immunocytochemistry Louise Badruna, Vincent Burlat, and Cộdric Y Montanier 15 Determining theLocalization ofCarbohydrate Active Enzymes Within Gram-ưNegative Bacteria Richard McLean, G DouglasInglis, Steven C Mosimann, Richard R.E Uwiera, and D WadeAbbott 16 Analysis ofComplex Carbohydrate Composition inPlant Cell Wall Using Fourier Transformed Mid-Infrared Spectroscopy (FT-IR) Ajay Badhan, Yuxi Wang, and Tim A McAllister 17 Separation andVisualization ofGlycans by Fluorophore-ưAssisted Carbohydrate Electrophoresis Mộlissa Robb, Joanne K Hobbs, and Alisdair B Boraston 18 A Rapid Procedure forthePurification of8-Aminopyrene Trisulfonate (APTS)-Labeled Glycans forCapillary Electrophoresis (CE)-Based Enzyme Assays Hayden J Danyluk, Leona K Shum, and Wesley F Zandberg 169 181 199 209 215 223 Part IV CAZyme Structure, Discovery, and Prediction Methods 19 Probing theComplex Architecture ofMultimodular Carbohydrate-Active Enzymes Using aCombination ofSmall Angle X-Ray Scattering andX-Ray Crystallography Mirjam Czjzek and Elizabeth Ficko-Blean 20 Metagenomics andCAZyme Discovery Benoit J Kunath, Andreas Bremges, Aaron Weimann, Alice C McHardy, and Phillip B Pope 21 Identification ofGenes Involved intheDegradation ofLignocellulose Using Comparative Transcriptomics Robert J Gruninger, Ian Reid, Robert J Forster, Adrian Tsang, and Tim A McAllister 22 Isolation andPreparation ofExtracellular Proteins fromLignocellulose Degrading Fungi forComparative Proteomic Studies Using Mass Spectrometry Robert J Gruninger, Adrian Tsang, and Tim A McAllister 239 255 279 299 Index 309 Contributors D.WadeAbbott Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada; Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada JaneWittrupAgger Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark GregoryArnal Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada Magnusỉ.Arntzen Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, s Akershus, Norway LiorArtzi Faculty of Biochemistry, Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel JathavanAsohan Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada MohamedA.Attia Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; Department of Chemistry, University of British Columbia, Vancouver, BC, Canada AjayBadhan Lethbridge Research and Development Centre, Agricultures and Agri-Food Canada, Lethbridge, AB, Canada LouiseBadruna LISBP, Universitộ de Toulouse, CNRS, INRA, INSA, Toulouse, France EdwardA.Bayer Faculty of Biochemistry, Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel AlisdairB.Boraston Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, USA AndreasBremges Computational Biology of Infection Research, Helmholtz Centre for Infection Research, Braunschweig, Germany; German Center for Infection Research (DZIF), Braunschweig, Germany HarryBrumer Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; Department of Chemistry, University of British Columbia, Vancouver, BC, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada VincentBurlat Laboratoire de Recherche en Sciences Vộgộtales, UMR 5546 UPS/ CNRS, Castanet-Tolosan, France DarrellCockburn Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA DanielJ.Cosgrove Department of Biology, Pennsylvania State University, University Park, PA, USA MirjamCzjzek UPMC Univ Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Sorbonne Universite, Roscoff, Bretagne, France HaydenJ.Danyluk Simon Fraser University, Department of Molecular Biology and Biochemistry, Burnaby, BC, Canada ix x Contributors ClaireDumon LISBP, Universitộ de Toulouse, CNRS, INRA, INSA, Toulouse, France DanielM.Durachko Department of Biology, Pennsylvania State University, University Park, PA, USA VincentG.H.Eijsink Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, s Akershus, Norway ElizabethFicko-Blean UPMC Univ Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Sorbonne Universite, Roscoff, Bretagne, France CarlosM.G.A.Fontes CIISA Faculdade de Medicina Veterinỏria, Universidade de Lisboa, Lisbon, Portugal RobertJ.Forster Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada JulieM.Grondin Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada RobertJ.Gruninger Lethbridge Research and Developmental Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada NathanK.Hepler Department of Biology, Pennsylvania State University, University Park, PA, USA Joanne K Hobbs Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada G.DouglasInglis Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada DarrylR.Jones Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada Benoit J.Kunath Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, s, Norway DavidN.Langelaan Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada GalinaMai-Gisondi Department of Bioproducts and Biosystems, Aalto University, Espoo, Aalto, Finland EmmaR Master Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada TimA.McAllister Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada NicholasMcGregor Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; Department of Chemistry, University of British Columbia, Vancouver, BC, Canada Alice C.McHardy Computational Biology of Infection Research, Helmholtz Centre for Infection Research, Braunschweig, Germany LaurenS.McKee Division of Glycoscience, School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden RichardMcLean Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada; Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada CộdricY.Montanier LISBP, Universitộ de Toulouse, CNRS, INRA, INSA, Toulouse, France Transcriptomic Identification of Genes Involved in Lignocellulose Degradation 297 consortium in 2011 [16] Users are also encouraged to consult the expertise of your service provider to identify the most appropriate approach 26 Adapters/Illumina_TruSeq_mRNA_adaptors.fa should contain the sequences of the adapters used in the RNA-Seq library construction, in FASTA format; the -t parameter should be no more than the number of available processor cores 27 SORTMERNA should be set to the directory where SortMeRNA is installed 28 This command requires at least 30GB of available RAM 29 The k-max parameter should be an odd number at least less than the read length; the -t parameter should be no more than the number of available processor cores 30 A summary of the transcriptome quality will be output in evaluation/transrating/assemblies.csv and quality statistics for each transcript will be output in evaluation/transrating/transcripts/contigs.csv 31 Set BUSCO to the directory where BUSCO is installed Set the -l parameter to an appropriate taxonomic group for which you have installed BUSCO data files A summary of the results will be saved in evaluation/run_BUSCO-OGS/ short_summary_BUSCO-OGS 32 The functional significance of these differentially expressed transcripts can be assessed following functional annotation of the sequences References Tsang A, Butler G, Powlowski J, Panisko EA, Baker SE (2009) Analytical and computational approaches to define the Aspergillus niger secretome Fungal Genet Biol 46:S153S160 Kolbusz MA, Di Falco M, Ishmael N, Marqueteau S, Moisan MC, da Silva BC, Powlowski J, Tsang A (2014) Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila Fungal Genet Biol 72:1020 Gruninger RJ, Puniya AK, Callaghan TM, Edwards JE, Youssef N, Dagar SS, Fliegerova K, Griffith GW, Forster R, Tsang A, McAllister T, Elshahed MS (2014) Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential FEMS Microbiol Ecol 90(1):117 Couger MB, Youssef NH, Struchtemeyer CG, Liggenstoffer AS, Elshahed MS (2015) Transcriptomic analysis of lignocellulosic bio- mass degradation by the anaerobic fungal isolate Orpinomyces sp strain C1A.Biotechnol Biofuels 8:208 Solomon KV, Haitjema CH, Henske JK, Gilmore SP, Borges-Rivera D, Lipzen A, Brewer HM, Purvine SO, Wright AT, Theodorou MK, Grigoriev IV, Regev A, Thompson DA, OMalley MA (2016) Early-ư branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes Science 351(6278):11921195 Pollegioni L, Tonin F, Rosini E (2015) Lignin-ư degrading enzymes FEBS J282(7): 11901213 Song L, Florea L (2015) Rcorrector: efficient and accurate error correction for Illumina RNA-seq reads GigaScience 4:48 Jiang H, Lei R, Ding SW, Zhu S (2014) Skewer: a fast and accurate adapter trimer for next-generation sequencing paired-end reads BMC Bioinformatics 15:182 298 Robert J Gruninger et al 9 Kopylova E, Noe L, Touzet H (2012) SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data Bioinformatics 28:32113217 10 Crusoe MR, Alameldin HF, Awad S etal (2015) The khmer software package: enabling efficient nucleotide sequence analysis F1000 Research 4:900 11 Li D, Liu CM, Luo R, Sadakane K, Lam TW (2015) MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph Bioinformatics 31:16741676 12 Smith-Unna RD, Boursnell C, Patro R, Hibberd JM, Kelly S (2015) TransRate: refer- ence free quality assessment of de-novo transcriptome assemblies bioRxiv 021626 13 Simao FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM (2015) BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs Bioinformatics 31:32103212 14 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNAseq data with DESeq2 Genome Biol 15:550 15 Wolfe R (2011) Techniques for cultivating methanogens Methods Enzymol 494:122 16 Encode consortium https://genome.ucsc edu/ENCODE/protocols/dataStandards/ ENCODE_RNAseq_Standards_V1.0.pdf Chapter 22 Isolation andPreparation ofExtracellular Proteins fromLignocellulose Degrading Fungi forComparative Proteomic Studies Using Mass Spectrometry RobertJ.Gruninger, AdrianTsang, andTimA.McAllister Abstract Fungi utilize a unique mechanism of nutrient acquisition involving extracellular digestion To understand the biology of these microbes, it is important to identify and characterize the function of proteins that are secreted and involved in this process Mass spectrometry-based proteomics is a powerful tool to study complex mixtures of proteins and understand how the proteins produced by an organism change in response to different conditions Many fungi are efficient decomposers of plant cell wall, and anaerobic fungi are well recognized for their ability to digest lignocellulose Here, we outline a protocol for the enrichment and isolation of proteins secreted by anaerobic fungi after growth on simple (glucose) and complex (straw and alfalfa hay) carbon sources We provide detailed instruction on generating protein fragments and preparing these for proteomic analysis using reversed phase chromatography and mass spectrometry Key words Exo-proteome, Fungi, CAZy, Proteomics, Lignocellulose, Mass spectrometry Introduction The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well recognized and in recent years has received renewed interest [1, 2] Aerobic fungi utilize powerful oxidative enzymes to break down lignin and expose cellulose and hemicellulose [3] This mechanism is not available to anaerobic fungi and these microbes have likely evolved a unique approach for breaking lignin-carbohydrate bonds Extensive genomic and transcriptomic efforts have been undertaken and this work has revealed that these fungi utilize a large repertoire of carbohydrate-active enzymes (CAZY enzymes) to degrade the plant cell wall [46] Many of these enzymes show low levels of sequence identity to proteins that have been characterized to date [46] Fungi obtain nutrients from their environment by secreting a potent mixture of enzymes that digest carbohydrates, proteins, and lipids D Wade Abbott and Alicia Lammerts van Bueren (eds.), Protein-Carbohydrate Interactions: Methods and Protocols, Methods in Molecular Biology, vol 1588, DOI10.1007/978-1-4939-6899-2_22, â Springer Science+Business Media LLC 2017 299 300 Robert J Gruninger et al Identifying and characterizing secreted proteins is essential to fully understand the biology of these microbes Proteomics is a powerful tool to study complex mixtures of proteins and understand how the proteins expressed by an organism change in response to different conditions The proteome is an inherently complex system to analyze due to its dynamic nature The composition of the proteome changes and is dependent on a number of factors such as cell cycle stage, metabolic state, and environmental conditions Selecting a method that enables specific fractions of the proteome to be targeted for analysis is crucial to the design of successful proteomics experiments A combination of computational and experimental tools can be used to predict and identify secreted proteins The commonly used bioinformatic program SignalP predicts whether a protein is secreted by identifying the presence of an N-terminal secretory signal peptide [7] One of the primary drawbacks to this approach is the lack of selectivity in distinguishing extracellular proteins from resident proteins of the Golgi apparatus and endoplasmic reticulum Consequently, many false positives may be generated when bioinformatically predicting extracellular proteins [8] Proteomics studies examining the soluble extracellular fraction of microbial cultures by protein mass spectrometry can be used to provide strong direct evidence that detected proteins are indeed extracellular A key aspect of proteomic identification of proteins is the ability to search peptide fingerprints against a database of known proteins This requires the organism being studied, or a close homologue, to have a well-annotated genome and/or transcriptomes that can serve as this database In this chapter, we will provide a detailed protocol for qualitatively examining what effect varying the carbon source used to grow lignocellulose degrading fungi has on the composition of the exoproteome We will specifically provide information on the growth of cultures, isolation and concentration of secreted proteins, in solution trypsin digestion of proteins, and preparation of peptides for mass spectrometry analysis (Fig 1) We will not go into step-by-step details of results interpretation as this analysis area is study dependent and there are a number of approaches that can be applied Our laboratories work on the biochemical mechanisms of plant cell wall digestion in the phylum Neocallimastigomycota and these cultures will be used specifically as an example of proteome analysis However, it should be noted that this approach can be used to examine the exo-proteome of any microbe grown in liquid culture Materials 2.1 Cell Culture Lowes semidefined anaerobic media with appropriate carbon source Vacuum filter fitted with Bỹchner funnel Isolation and Preparation of Extracellular Proteins for Comparative Proteomic Studies 301 Fig Representation of the steps involved in the identification of proteins that are secreted during growth of fungi in liquid culture using a mass spectrometry-based proteomics approach Whatmann quantitative 50 fast flow filter paper Microfuge 2.0mL Eppendorf tubes 2.2 Isolation andEnrichment ofSecreted Proteins SDS-PAGE gel and electrophoresis apparatus Sigma Brilliant Blue G (other Coomassie reagents for staining of total protein from other manufacturers can be substituted) Biorad RC DC Protein assay kit (other methods for quantification of total protein or Bradford reagent from other manufacturers can be substituted) 100% (w/v) trichloroacetic acid (TCA)prepare solution by adding 340mL of d2H2O to 500g TCA (see Note 1) Acetone (Mass spectrometry grade) 100mM Ammonium bicarbonate (NH4HCO3), pH8.5to make solution dissolve 7.906g of NH4HCO3 in 950mL of d2H2O, adjust pH to 8.5 and top up volume to 1000mL Narrow range pH paper (pH range 8.09.0) 2.3 Alkylation andTrypsin Digestion 0.1% (w/v) Anionic Acid Labile Surfactant (AALS) II in 100mM NH4HCO3, pH8.5to make solution dissolve 5mg of AALS II in 5mL of previously prepared 100mM NH4HCO3, pH8.5 100mM dithiothreitol (DTT) in 100mM NH4HCO3, pH8.5to make solution dissolve 1.54g of DTT in 100mL of previously prepared 100mM NH4HCO3, pH8.5 302 Robert J Gruninger et al 500mM Iodoacetamide in 100mM NH4HCO3, pH8.5to make solution dissolve 0.925g of Iodoacetamide in 10mL of previously prepared 100mM NH4HCO3, pH8.5 100ng/L trypsin (mass spectrometry grade) in 50mM acetic acid (add 0.0287mL of glacial acetic acid to 9.97mL of d2H2O to make 50mM acetic acid solution) (see Note 2) 1% (v/v) Trifluoroacetic acid (TFA)add 1mL of sequencing grade TFA to 99mL of d2H2O to make 100mL of 1% TFA 2.4 Peptide Enrichment andSolvent Exchange C18 ZipTips (EMD Millipore) 2.5 Preparation ofAnaerobicMedia Prepare enough liquid modified Lowes media supplemented with 1% w/v carbon source (1g carbon/100mL media) to make 3ì100mL media bottles per carbon source (see Table 1) Combine media components as indicated in Table and add water to a final volume of 950mL.Add 50mL 8% w/v Na2CO3 to make final volume of media 1L Acetonitrile (mass spectrometry grade) 0.1% (v/v) formic acid (FA), 5% (v/v) acetonitrileadd 1mL of 1% TFA, 0.5mL of acetonitrile to 8.5mL of d2H2O to make 10mL of solvent Using a Hungate system (see Note 3), gently bubble reduced, anaerobic CO2 into the media and bring media to a boil being careful that the media does not boil over At 0.1g of cysteine to boiling media to fully reduce it Boil the media until it changes color from pink to a pale yellow/clear Dispense 100mL of reduced media (see Note 4) into culture bottle containing the 1g of appropriate carbon source to make the final concentration 1% (w/v for solid substrates or v/v for liquid substrates) Cap bottles containing reduced media and carbon source and autoclave (see Note 5) Methods 3.1 Growth ofFungal Cultures Grow anaerobic fungi using aseptic technique under strict ưanaerobic conditions When conducting comparative experiments it is important to have at least two biological replicates to allow statistical analysis to be carried out If possible, three biological replicates are advised This experimental design can be used for comparative analysis of the exo-proteome of anaerobic fungi grown using three different carbon sources: (1) glucose, (2) barley straw, (3) alfalfa hay (see Note 6) To determine basal protein secretion 3g CaCl2 8.1g NH4Cl 10mL Volatile fatty acid solution 10mL 0.1% w/v heminc 0.2g nicotinamide 0.2g Calcium-d-ưpanthothenate 0.25g 1,4-naphthoquinone 0.025g cyanocobalamin 0.025g para-ưaminobenzoic acid 0.05g NH4VO3 0.025g ZnCl2 0.025g CuCl2-ư2H2O 1g tryptone peptone 0.5g yeast extract 1.5g PIPES buffer b a Trace mineral solution must be made using 0.2M HCl to dissolve components Volatile fatty acids are added to 700mL of 0.2M NaOH and the pH is adjusted to 7.5 with 1M NaOH.The solution is then diluted with water to a final volume of 1L c Hemin is dissolved in 0.005M NaOH at a concentration of 0.1% w/v d Resazurin is dissolved in water at a concentration of 0.1% w/v 0.025g folic acid 0.025g biotin 0.07g NaSeO3 0.2g pyridoxine-HCl 10mL vitamin mix 0.55mL n-valeric acid 0.2g thiamin 0.05g CoCl2-ư6H2O 0.47mL isovaleric acid 0.55mL 2-methylbutyric acid 0.2g riboflavin 1.85mL butyric acid 3mL Propionic acid 6.85mL Acetic acid Vitamin mix 1mL 0.1% w/v resazurind 0.2g FeSO4-ư7H2O 0.25g H3BO3 7.5g MgSO4-ư7H2O 0.25g NaMoO4-ư2H2O 10mL Trace minerals solution 0.25g NiCl2-ư6H2O 9g NaCl 0.25g MnCl2-ư4H2O Trace minerals solutiona Volatile fatty acid solutionb 55mL macronutrient solution 4.5g KH2PO4 9g KCl 75mL PO4 solution Macronutrient solution PO4 solution Lowes semidefined media (per 1L) Table Components for preparing 1L of modified Lowes semidefined anaerobic media without clarified rumen fluid or carbon source Isolation and Preparation of Extracellular Proteins for Comparative Proteomic Studies 303 304 Robert J Gruninger et al by fungi, grow three cultures using glucose as the sole carbon source To identify proteins that are secreted by the fungus to break down the plant cell wall, grow three cultures with barley straw and three cultures with alfalfa hay provided as the sole carbon source (see Note 7) When media has cooled, inoculate cultures with fungal mycelia and incubate at 39C under anaerobic conditions for 72h Do not shake flasks (see Note 8) Using a vacuum filter fitted with a Bỹchner funnel and Whatmann quantitative 50 fast flow filter paper (GE Lifesciences, Mississauga ON), carefully separate the liquid culture from the fungal mycelia and any insoluble material (see Note 9) Further clarify the supernatant by centrifugation at 12,000 15,000ìg for 15min at 4C.This cell-free culture supernatant contains the fungal proteins secreted during growth Carefully transfer supernatant without disturbing pellet and make several 1.5 mL tubes containing clarified cell-free culture supernatant Freeze any tubes that will not be used right away for protein isolation which can be used as backups, technical replicates, or for additional experiments 3.2 Isolation andEnrichment ofSecreted Proteins Contamination of samples with keratin can be minimized by always wearing gloves when handling material and samples, working in a laminar flow hood, using a dedicated set of pipettes and pipette tips, and filtering solutions when possible Most detergents should be avoided as these can interfere with sample ionization and are difficult to remove from samples All solutions should be freshly prepared using HPLC grade reagents and regularly checked to ensure they not contain visible particles or fibers Quantify the total protein concentration in the supernatant using the RC DC protein assay kit (Biorad, Mississauga, ON) as described by the manufacturer It is important to know how much protein is in your sample so that the correct amount of protease can be used to fragment the protein into peptides Run an SDS-PAGE gel to visually inspect the presence of protein bands after Coomassie staining the gel If no protein bands are visible on the SDS-PAGE gel, concentrate proteins by TCA precipitation 3.3 TCA Precipitation ofProteins inSolution: Reductive Alkylation ofFree Cysteines Mix one volume of 100% (w/v) TCA to four volumes of clarified cell-free culture supernatant and incubate for 10min at 4C A white precipitate should form Pellet precipitated protein by centrifugation at 1015,000ìg for 10min Decant supernatant Wash pellet with 500L of chilled acetone (20C) Break up the pellet by pipetting up and down Isolation and Preparation of Extracellular Proteins for Comparative Proteomic Studies 305 Pellet protein by centrifugation at 1015,000ìg for 10min Decant supernatant Repeat steps and two more times for a total of three acetone washes Dry pellet in fume hood for 3060min Residual acetone will reduce the ability to redissolve protein pellet Dissolve pellet in 0.1% (W/V) AALS II in 100mM NH4HCO3 pH8.5 Solubilizing TCA precipitated protein can be difficult and conditions may need to be optimized for different samples 3.4 Reductive Alkylation ofFree Cysteines Resuspend an appropriate amount of sample material (5g of total protein) in 100mM NH4HCO3, pH8.5 buffer Check that protein sample is between pH8.0 and 8.5 by spotting a small amount on pH paper and adjust if necessary with 1.0M NH4HCO3 Add an appropriate amount of AALS II solution to achieve a final concentration of 0.1% This detergent facilitates trypsin digestion by denaturing proteins and has the advantage of not interfering with the subsequent LC-MS/MS analysis since it is broken down in acidic conditions Add the 100mM DTT stock solution to samples to achieve a final concentration of 10mM DTT (e.g., 10L of 100mM DTT per 90L of sample) Incubate at room temperature for 30min This will reduce disulfide bonds in the protein Add the 500mM iodoacetamide stock solution to fully reduced samples to achieve a final concentration of 50mM (e.g., add 10L per 90L of reduced sample) Incubate in the dark at room temperature for 30min Adjust concentration of DTT to 50mM to quench remaining iodoacetamide 3.5 Trypsin Digestion ofProtein Digest proteins into peptides by adding trypsin at a ratio of 1:50 trypsin:protein Vortex sample to mix and incubate with gentle agitation at 37C for 418h (see Note 10) Stop protease cleavage by adding 510L of 1% (v/v) TFA to decrease the pH of sample to 23 Leave at room temperature for at least 30min to allow for the degradation of the AALS II (see Note 11) 3.6 Sample Desalting andBuffer Exchange Using C18 Zip-Tip Prior to analysis of samples by mass spectrometry, all salts must be removed and the sample placed in a mass spectrometry-compatible solvent system Several chromatography based commercial systems are available to quickly desalt and buffer exchange protein samples We will describe the use of ZipTips (EMD Millipore, Etobicoke, ON) for this purpose ZipTips are pipette tips containing a small 306 Robert J Gruninger et al amount of C18 resin in the tip with binding capacity of up to 5g of protein A similar product called Pierce C18 tip is available from Pierce (ThermoFishcer Scientific, Mississauga, ON) and has a binding capacity of 880g and can be used for samples with larger amounts of protein Place ZipTip on a standard 10-L pipettor Single-channel and multi-channel pipettors are compatible and either can be used to accommodate the required throughput for a particular experiment To bind sample, aspirate into ZipTip and pipette up and down several times The protein/peptides will bind to the resin in the tip while the buffer salt components will not To exchange buffers, aspirate the ZipTip with bound protein/ peptides into the mass spectrometry-compatible solvent Wash the resin with several pipette tip volumes to fully exchange solvent Elute the resin-bound sample material directly into a fresh tube with a volume of high organic solvent Typically, 80% Acetonitrile: 0.1% Formic acid (v/v) is used as eluent The sample will be eluted from the resin in a volume of 14L Adjust the volume of the eluent solution such that the final acetonitrile concentration is no more than 5% (v/v) If LC-MS/MS involves reversed phase separation of peptide on a C18 column, a suitable solvent system is 0.1% (v/v) formic acid, 5% (v/v) acetonitrile (see Note 12) The sample is now ready for analysis by mass spectrometry (see Note 13) Notes Always use personal protective equipment when working with TCA as it can cause chemical burns Pierce Trypsin Protease (ThermoFischer Sci) is recommended This trypsin is highly purified, free from chymotrypsin, and has been chemically modified to enhance its stability and prevent autolysis during protease digestion The Hungate system is a specialized apparatus for preparing anaerobic media and can be used to enable researchers to work with anaerobic cultures and media on the bench-top while maintaining strict anaerobic conditions Readers are referred to the review by Wolfe for details of setting up and working with a Hungate system [9] Care must be taken to ensure that the media remains anaerobic Resazurin is included in the media to serve as an oxygen indicator The media will change color from pale yellow/clear to pink if oxygen has been introduced into the media Isolation and Preparation of Extracellular Proteins for Comparative Proteomic Studies 307 Some carbon sources such as glucose will caramelize if autoclaved for too long or at too high of temperature Be sure to set the autoclave cycle appropriately to prevent this It is also important to secure the cap on bottle to prevent it from coming off during autoclave cycle Barley is a dicot and alfalfa is a monocot so this experiment will also provide an opportunity to examine whether these fungi utilize unique mechanisms to degrade the cell wall of monocot and dicot plants The growth of cultures on glucose, or another simple sugar such as fructose, is used to determine baseline protein production and to serve as a reference to identify proteins that are produced to digest the plant cell wall The complex carbon sources included in the study can be substituted to address the particular goals of different end-users It is essential that cultures be grown under the same conditions, and are harvested at the same growth stage It is also important to be gentle when growing fungal cultures and preparing cell-free culture supernatant to minimize the chance of cell lysis and leakage of intracellular proteins into the media Transfer fungal mycelia into a separate falcon tube and freeze in liquid nitrogen Retain this material for RNA isolation which can subsequently be used for comparative transcriptomic studies 10 Trypsin is optimally active at pH8.0 and it is advisable to check that the pH of your sample is in this range by spotting a small volume on pH paper The ratio of protease to sample and time of digestion may need to be optimized if digestion is incomplete 11 Trypsin digestion is only one approach for fragmenting proteins prior to mass spectroscopy Other proteases such as LysC, chymotrypsin, AspN or chemicals such as cyanogen bromide can also be used to generate peptides [10] 12 Prior to injection of peptides into the mass spectrometer, peptides are bound to a C18 reversed phase column in an aqueous buffer and eluted using a linear gradient of increasing acetonitrile To maximize peptide retention to the column, it is important to minimize the organic solvent content of the sample 13 The protocol outlined here provides users with the tools to isolate the proteins secreted by a microbe when grown on a particular carbon source This protocol can be applied to other experimental questions aimed at examining the effect of many growth variables on protein production in microbes For most users, mass spectrometry analysis of samples will be carried out by commercial service providers, core facilities, or collaborating labs with expertise in proteomics There are a range of approaches for the analysis of proteomic data and users are 308 Robert J Gruninger et al encouraged to consult their service providers or collaborator to determine the most appropriate approach to sample analysis The interpretation of results and their relevance to a particular biological system is study-dependent and should be validated through additional experimentation using complementary approaches References Gruninger RJ, Puniya AK, Callaghan TM, Edwards JE, Youssef N, Dagar SS, Fliegerova K, Griffith GW, Forster R, Tsang A, McAllister T, Elshahed MS (2014) Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential FEMS Microbiol Ecol 90(1):117 Haitjema CH, Solomon KV, Henske JK, Theodorou MK, O'Malley MA (2014) Anaerobic gut fungi: advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production Biotechnol Bioeng 111(8):14711482 Youssef NH, Couger MB, Struchtemeyer CG, Liggenstoffer AS, Prade RA, Najar FZ, Atiyeh HK, Wilkins MR, Elshahed MS (2013) The genome of the anaerobic fungus Orpinomyces sp strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader Appl Environ Microbiol 79:46204634 Couger MB, Youssef NH, Struchtemeyer CG, Liggenstoffer AS, Elshahed MS (2015) Transcriptomic analysis of lignocellulosic biomass degradation by the anaerobic fungal isolate Orpinomyces sp strain C1A.Biotechnol Biofuels 8:208 Solomon KV, Haitjema CH, Henske JK, Gilmore SP, Borges-Rivera D, Lipzen A, Brewer HM, Purvine SO, Wright AT, Theodorou MK, Grigoriev IV, Regev A, Thompson DA, O'Malley MA (2016) Early-ư branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes Science 351(6278):11921195 Pollegioni L, Tonin F, Rosini E (2015) Lignin-ư degrading enzymes FEBS J282(7): 11901213 Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions Nat Methods 8:785786 Tsang A, Butler G, Powlowski J, Panisko EA, Baker SE (2009) Analytical and computational approaches to define the Aspergillus niger secretome Fungal Genet Biol 46:S153S160 Wolfe R (2011) Techniques for cultivating methanogens Methods Enzymol 494:122 10 Swaney DL, Wenger CD, Coon JJ (2010) Value of using multiple proteases for large-scale mass spectrometry-based proteomics JProteome Res 9(3):13231329 Index A Acetylated xylo-oligosaccharides45 Acetyl xylan esterase (AcXE)4554 Affinity electrophoresis119126 Aldonic acids 72, 74, 75, 77, 78, 8384, 8790 8-Aminonaphthalene-1,3,6-trisulphonic acid (ANTS) 215, 216, 218220 Assembly 15, 94, 96, 98, 101, 109110, 169178, 239, 262267, 281, 290292 B Binding studies 94, 104105 Binning 263, 265267 Bioinspired169178 C Capillary electrophoresis (CE)224234 Carbohydrate active enzymes (CAZymes) 15, 37, 45, 59, 200, 203207, 216, 239, 240, 242251, 255, 259, 266, 269272, 274, 280, 299 Carbohydrate binding modules (CBMs) 94, 95, 102, 103, 108, 110, 111, 119, 124, 125, 129140, 146, 147, 155, 181187, 189, 190, 193, 194, 239, 240, 248, 269, 270 Carbohydrates 3, 17, 35, 37, 59, 79, 93, 119, 146, 182, 209, 215, 226, 239, 255, 280, 299 Cell fractionation201 Cellulases77 Cellulose7277, 82, 8788, 93, 98, 100102, 104, 108, 109, 121, 130, 131, 134136, 139, 140, 157, 158, 169, 181183, 191, 209, 240, 255, 271, 274, 280, 299 Cellulosomes9398, 100105, 108110, 112, 114, 183, 270 Chemical shift perturbations150151 Cohesin94, 95, 9798, 102110, 113, 241245, 247, 248, 270 Colorimetric assay 47, 50, 60, 61 Copper-bicinchoninic acid (BCA)313 Creep 157162, 164 D Diffusion 170, 172, 174175, 224, 233, 234 3,5-Dinitrosalicylic acid (DNSA)2735 Dissect and build 240242, 249 Dissociation constants (Kd)124, 130, 134, 136, 139, 144146, 150152 Dockerin 94, 95, 9798, 100111, 113, 241245, 247, 248, 270 E Electrophoresis98, 105, 108, 113, 119126, 215, 218219, 226, 229, 232, 234, 301 Enzymatic hydrolysis27 Enzyme assays 5, 8, 10, 12, 18, 110, 224, 226234 Enzyme kinetics 16, 18, 2735, 60 Enzymes 5, 16, 27, 38, 45, 59, 71, 93, 119, 169, 182, 199, 216, 224, 239, 255, 299 Enzymology 313, 1524, 2736, 37, 38, 4043, 45, 4855, 5969, 71, 79, 86, 9395, 105, 107111, 114, 119-125, 169, 182, 183, 199207, 216, 218, 219, 223234, 239251, 255, 268, 269, 272, 274, 280, 295, 299 Exo-proteome 300, 302 Expansins 157159, 162164 F Filter paper 158, 159, 163, 274, 280, 283, 301, 304 Fluorescence63, 64, 67, 129, 130, 134136, 140, 169, 172, 174, 175, 177, 178, 189, 190, 192, 195, 219, 281 Fluorescence quenching 136, 137, 140, 177 Fluorescence recovery after photobleaching (FRAP)169178 Fluorescent label183 Fluorophore130, 136, 170, 172173, 177, 178, 215, 216, 218220, 224226 Fluorophore-assisted carbohydrate electrophoresis (FACE) 215220, 225232, 234 Fourier transformed infrared spectroscopy209214 4-Methylumbelliferyl acetate (4MUA)45, 46, 49, 52, 54 Fungi102, 158, 280, 282284, 294, 295, 299307 G Gemdiols75, 78 Glycan digestion 15, 215, 216, 218220, 224, 226234 Glycan purification 225, 231232 Glycosidases3, 16, 224, 226, 228 D Wade Abbott and Alicia Lammerts van Bueren (eds.), Protein-Carbohydrate Interactions: Methods and Protocols, Methods in Molecular Biology, vol 1588, DOI10.1007/978-1-4939-6899-2, â Springer Science+Business Media LLC 2017 309 Protein-Carbohydrate Interactions: Methods and Protocols 310 Index Glycoside hydrolase (GH) 313, 1524, 7778, 216, 217, 227, 239, 241, 269 Glycosyltransferase5969 Gram-negative bacteria 200, 203207 Graphite 231, 232, 234 H Heteronuclear single quantum coherence (HSQC) 143146, 150152 High performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-ưPAD) 1621, 23, 83 High performance liquid chromatography (HPLC) 1620, 47, 50, 53, 59, 62, 64, 6768, 8183, 216, 244, 304 Hydrolysis 12, 21, 23, 27, 31, 34, 35, 45, 60, 90, 112, 169, 228, 234 Hydrophilic interaction liquid chromatography (HILIC) 72, 81, 83 I Immunocytochemistry181187, 189, 190, 193, 194 Interactions 61, 72, 94, 119, 129, 143, 158, 169, 183, 205, 226, 239, 265 K Kd (dissociation constant) 124, 130, 134, 136, 139, 144146, 150152 Kinetics7, 1012, 1524, 2735, 37, 39, 4144, 46, 5969, 76 L Ligands43, 119, 124, 125, 130, 133, 134, 136, 137, 139141, 143146, 152, 153, 169, 182, 183, 240, 242, 248, 251 Lignins121, 181, 191, 209, 211, 213, 280, 299 Lignocellulose 110, 182, 255, 271, 280281, 283296, 299302, 304307 Lipid II transfer 62, 64, 6768 Lytic polysaccharide monooxygenase 71, 72, 7490 M Malachite green dye 5961, 64, 6669 Mass spectrometry 71, 72, 7490, 100, 110, 299302, 304307 Meta-genomics267 Metal-dependent 37, 39, 4144 Microbial communities 263, 266, 270, 273 Microscale thermophoresis (MST)129140 Microscopy 88, 169, 182, 183, 185189, 240 Multi-enzymatic complex 93, 183 Multimodular93, 239, 240, 242251, 270 N -Naphthyl acetate45, 46, 49, 5152, 54 Neocallimastigomycota280, 299, 300 Nuclear magnetic resonance (NMR) 47, 48, 55, 79, 143155 O Oligosaccharides3, 12, 1623, 35, 45, 46, 49, 52, 72, 74, 76, 77, 79, 8384, 88, 89, 215, 216, 219, 271 Osmotic shock 200202, 204 P Para-nitrophenol (pNP) 4951, 53, 59, 60, 6566 Pectate 3744, 182, 183 Pectins37, 157, 173, 181, 209, 211, 271 Plant cell wall37, 38, 93, 103, 157159, 162, 164, 169178, 181187, 189, 190, 193, 194, 209214, 280, 294, 299, 300, 304, 307 p-nitrophenyl acetate (pNP-acetate) 45, 46, 4851, 53, 54 Polyacrylamide gel electrophoresis (PAGE) 119, 218 Polysaccharide 3, 8, 12, 35, 37, 46, 49, 71, 72, 7490, 93, 119126, 129, 139, 157, 169, 182, 216, 217, 219, 256, 270, 273 Polysaccharide lyases (PLs)15, 37, 38, 4043, 144, 145, 239, 269 Porous graphitized carbon (PGC) 75, 76, 8184, 89, 225, 226, 231, 232 Protein/carbohydrate interactions 129, 143155, 169178, 183, 248 Proteins3, 15, 27, 38, 60, 96, 119, 130, 143, 157, 169, 181, 199, 210, 215, 224, 239, 267, 280, 299 Proteomics 95, 294, 299302, 304307 R Reducing sugars 3, 5, 12, 2731, 33, 35, 99, 110, 111, 224 RNA sequencing (RNA-Seq) 284, 290, 295, 296 S Secretion302 Signal peptides199, 202, 203, 205, 206, 248, 300 Small angle X-ray scattering (SAXS)239251 Solid phase extraction (SPE) 225232, 234 Protein-Carbohydrate Interactions: Methods and Protocols 311 Index Structures15, 60, 96, 100, 137, 143, 146, 147, 150151, 153, 158, 170, 174, 177, 191, 201, 213, 224, 226228, 240243, 247, 248, 251, 258 Subcellular localization 199, 200, 203207 Surface binding sites119 T Transcriptomics279281, 283296, 299, 307 U Uronic acid37 UV/visible spectrophotometry 28, 31, 171 W Wall loosening proteins157 Wheat straw sections 183, 185, 187, 188, 190, 192, 193 Whole cell dot blot202205 X X-ray crystallography (XRC)239251 Xylan 31, 35, 4554, 80, 99, 112, 114, 121, 130, 182, 183, 194, 210, 211 Xylanase46, 48, 98, 106, 183 -Xylosidase-coupled assay50, 53 ... (Chapter 3) and Arnal etal (Chapter 1) in this volume), D Wade Abbott and Alicia Lammerts van Bueren (eds.), Protein-Carbohydrate Interactions: Methods and Protocols, Methods in Molecular Biology, ... van Bueren (eds.), Protein-Carbohydrate Interactions: Methods and Protocols, Methods in Molecular Biology, vol 1588, DOI 10.1007/978-1-4939-6899-2_1, â Springer Science+Business Media LLC 2017... multiple disciplines and holds vast promise for informing future innovations in renewable resource utilization and medicine Since the turn of the millennia, the field of protein-carbohydrate interactions