Determining the Structure of Oligosaccharides N‐ and O‐Linked to Glycoproteins

Louise Royle1, Raymond A. Dwek1, Pauline M. Rudd1

1 University of Oxford, Oxford
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.6
DOI:  10.1002/0471140864.ps1206s43
Online Posting Date:  March, 2006
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Abstract

Many proteins involved in biological events are glycosylated. A glycoprotein consists of a mixture of glycosylation variants of a single polypeptide chain, known as glycoforms. It has become clear that a detailed understanding of the roles which glycosylation plays in the biosynthesis, transport, biological function, and degradation of a glycoprotein can only be achieved when the protein and sugar(s) are viewed as an entity. Many glycoproteins can now be modeled by combining glycan sequencing data and oligosaccharide structural information with protein structural data. Pivotal to this approach is sensitive, state‐of‐the‐art oligosaccharide sequencing technology which can give a rapid insight into the glycosylation of a glycoprotein without the need for sophisticated equipment and expertise. This unit gives a detailed introduction into the analysis of glycans, and the many figures will help the user identify which type of experiment needs to be undertaken. Methods for releasing glycans from glycoproteins are followed by protocols for labeling and purifying (by HPLC) the glycans from the rest of the components. Strategies for N‐ and O‐glycan analysis are also included.

     
 
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Table of Contents

  • Glycan Analysis Complements Protein Structural Analysis to give a more Complete View of a Glycoprotein
  • Release of Glycans from Glycoproteins
  • Labeling the Released Glycans
  • Profiling by HPLC
  • Characterization of N‐ and O‐Linked Oligosaccharides
  • Basic Protocol 1: Enzymatic Release of N‐Linked Oligosaccharides by PNGase F Digestion in Solution
  • Basic Protocol 2: In‐Gel Enzymatic Release of N‐Linked Glycans by PNGase F from Proteins Separated in SDS‐PAGE Gel Bands
  • Basic Protocol 3: Identification/Confirmation of Glycoprotein by In‐Gel Trypsin Digestion
  • Basic Protocol 4: Manual Hydrazinolysis to Release N‐ and O‐Glycans
  • Basic Protocol 5: Fluorescent Labeling of the Glycan Pool with 2‐AB
  • Basic Protocol 6: Multidimensional Separation and Profiling of 2‐AB‐Labeled Glycans using NP‐, RP‐, and WAX‐HPLC
  • Support Protocol 1: Calibration of the HPLC System
  • Basic Protocol 7: Sequencing an Entire N‐Glycan Pool Simultaneously using Exoglycosidase Arrays
  • Basic Protocol 8: Sequencing O‐Glycans
  • Reagents and Solution
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enzymatic Release of N‐Linked Oligosaccharides by PNGase F Digestion in Solution

  Materials
  • Glycoprotein sample
  • Buffer A (see recipe)
  • Buffer B (see recipe)
  • 10% (w/v) dithiothreitol (DTT)
  • 1000 U/ml peptide N‐glycosidase F (PNGase F), recombinant glycerol‐free lyophilate (Roche Life Sciences)
  • Toluene
  • Sub‐boiling‐point‐distilled (SBPD) water
  • Vacuum centrifuge
  • 100°C temperature‐controlled heating block
  • Protein‐binding membrane (e.g., Micropure‐EZ centrifugal filter devices, Millipore.)
  • PCR tubes
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Basic Protocol 2: In‐Gel Enzymatic Release of N‐Linked Glycans by PNGase F from Proteins Separated in SDS‐PAGE Gel Bands

  Materials
  • 30% acrylamide/0.8% bis‐acrylamide solution (e.g., Protogel; National Diagnostics)
  • 0.5 M Tris base, pH 8.8
  • 1.5 M Tris base, pH 6.6
  • Sub‐boiling‐point‐distilled (SBPD) water
  • 10% (w/v) SDS
  • 10% (w/v) ammonium persulfate (APS)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED)
  • Glycoprotein sample
  • 0.5 M dithiothreitol (DTT)
  • 5× SDS sample buffer (see recipe)
  • 100 mM iodoacetamide
  • 25 mM Tris/190 mM glycine/0.5% (w/v) SDS
  • 50% (v/v) methanol/7% (v/v) acetic acid
  • 5% (v/v) methanol/7% (v/v) acetic acid
  • 20 mM NaHCO 3, pH 7.0
  • 1:1 (v/v) acetonitrile/20 mM NaHCO 3
  • 1000 U/ml peptide N‐glycosidase F (PNGase F) in 20 mM NaHCO 3
  • Acetonitrile
  • Dowex AG50X12 (H+ form; 100 to 200 mesh; Bio‐Rad), activated (see unit 12.7 for conditioned resin)
  • 1 M HCl
  • 70°C water bath or heating block
  • Roller‐mill‐type mixer (Spiramix from Denley Instruments, or equivalent)
  • Vacuum centrifuge
  • Sonicator
  • 0.45‐µm Millex‐LH/hydrophilic PTFE filter (Millipore)
  • 2.5‐ml syringe, lubricant‐free
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and Coomassie blue staining (unit 10.5)
NOTE: Iodoacetamide is light sensitive. It should be prepared fresh before each use.

Basic Protocol 3: Identification/Confirmation of Glycoprotein by In‐Gel Trypsin Digestion

  Materials
  • Gel pieces containing glycoprotein of interest
  • Acetonitrile
  • 20 and 50 mM ammonium hydrogen carbonate (NH 4HCO 3) in sub‐boiling‐point‐distilled (SBPD) water
  • 0.1 mg/ml sequencing‐grade trypsin (Roche Diagnostics) solution in SBPD water
  • 10% (v/v) formic acid in SBPD water
  • Vacuum centrifuge

Basic Protocol 4: Manual Hydrazinolysis to Release N‐ and O‐Glycans

  Materials
  • 0.01 to 2 mg/ml glycoprotein sample
  • Sub‐boiling‐point‐distilled (SBPD) or purified water
  • 0.1% (v/v) trifluoroacetic acid (TFA) in purified or SBPD water
  • Liquid nitrogen
  • Lyophilizer
  • Dry hydrazine (spec: ∼0.1% v/v H 2O by GC, measured on a 10 ft. × 1/4 in., column of Fluoropak/20% UCOW 550; thermal conductivity detector; Ashford et al., )
  • Argon
  • Anhydrous toluene
  • Acetic anhydride
  • 0.2 M sodium acetate
  • Dowex AG50X12 resin (H+ form; 200 to 400 mesh; Bio‐Rad)
  • 1 M HCl
  • 8:2:1 and 4:1:1 (v/v/v) butanol/ethanol/water
  • Flow dialyzer (use membrane with appropriate pore size for sample)
  • Hydrazinolysis tubes: 5‐ to 10‐ml glass tubes with screw thread suitable for a Miniert valve
  • Miniert valve (Pierce/Perbio Science)
  • Glass syringe with Teflon plunger and stainless‐steel needle
  • Acid‐washed long Pasteur pipets
  • Incubation equipment at 60° and 85°C
  • Vacuum pump and vapor trap
  • Poly‐Prep disposable chromatography columns (Bio‐Rad)
  • Rotary evaporator with clean glass tubes
  • 30‐mm wide Whatman 1 Chr chromatography paper
  • Pinking shears
  • Chromatography tank: 60 cm tall, equipped with solvent troughs and lid
  • Waxed paper
  • Lubricant‐free 2.5‐ml syringes
  • 0.45‐µm pore size, 13‐mm diameter hydrophilic PTFE Millex‐LCR filter (Millipore)

Basic Protocol 5: Fluorescent Labeling of the Glycan Pool with 2‐AB

  Materials
  • Glyko Signal 2‐aminobenzamide (2‐AB) labeling kit (Prozyme, GKK‐404) or LudgerTag 2‐AB labeling kit (LT‐KAB‐A2; http://www.ludger.com)
  • Glycoprotein sample
  • Sub‐boiling‐point‐distilled (SBPD) water
  • Acetonitrile
  • 200‐µl PCR tubes
  • 65°C incubator or heating block
  • GlycoClean S cartridges (Prozyme) or LudgerClean S cartridges (http://www.ludger.com) or 3MM chromatography paper (Whatman) cut into 10 × 3–cm pieces
  • 100‐ml glass beaker
  • Lubricant‐free 2.5‐ml syringes
  • 13‐mm diameter, 0.45‐µm pore size hydrophilic PTFE Millex‐LCR filters (Millipore)
  • Rotary evaporator and acid‐washed tubes
  • Vacuum centrifuge

Basic Protocol 6: Multidimensional Separation and Profiling of 2‐AB‐Labeled Glycans using NP‐, RP‐, and WAX‐HPLC

  Materials
  • Glycoprotein sample
  • 80% (v/v) acetonitrile
  • HPLC standards (see recipe): dextrose for NP‐HPLC, arabinose for RP‐HPLC, and fetuin N‐glycans for WAX‐HPLC
  • HPLC solvents appropriate for method:
    • Solvent A (see recipe; NP): 50 mM formate, adjusted to pH 4.4 with ammonia
    • Solvent B (NP): HPLC‐grade acetonitrile
    • Solvent C (see recipe; RP): 50 mM formate, adjusted to pH 5 with triethylamine
    • Solvent D (RP): 50:50 (v/v) solvent C/acetonitrile
    • Solvent E (see recipe; WAX): 500 mM formate, adjusted to pH 9 with ammonia
    • Solvent F (WAX): 10:90 (v/v) methanol/water
  • Waters Alliance 2695 Separations Module: combined autosampler, injector, degasser, and column heater
  • Waters 474, Waters 2475, or Jasco Fluorescence Detector (for 2‐AB, excitation: λ max 330 nm, band width 16 nm; emission λ max 420 nm, band width 16 nm)
  • Pentium PC
  • Waters Bus SAT/IN interface for data collection for Jasco detectors
  • Waters Bus LAC/E interface, internal board for PC
  • Column:
  • Normal phase: 4.6 × 250–mm TSKgel amide‐80 column (Anachem), GlycoSep N column (Prozyme), or LudgerSep N1 amide column (http://www.ludger.com); column temperature 30°C
  • Reversed phase: 4.6 × 150–mm, 3‐µm, 130‐Å pore‐size Hypersil ODS C18 column (Phenomenex) or GlycoSep R column (Prozyme); column temperature 30°C
  • WAX: Vydac protein WAX, 7.5 × 50–mm column (P/N 301 VHP 575) GlycoSep C column (Prozyme), or Ludger Sep C2 anion exchange column (http://www.ludger.com); column temperature ambient

Support Protocol 1: Calibration of the HPLC System

  Materials
  • N‐glycan pool (2‐AB labeled; see protocol 5)
  • Exoglycosidase enzymes and buffers (Table 12.6.4)
  • 200‐µl microcentrifuge tubes
  • Speedvac evaporator
  • 37°C incubator or water bath.
  • Micropure‐EZ centrifugal filter devices (Millipore)
  • Additional reagents and equipment for NP‐HPLC (see protocol 6)

Basic Protocol 7: Sequencing an Entire N‐Glycan Pool Simultaneously using Exoglycosidase Arrays

  Materials
  • Exoglycosidase enzymes (Table 12.6.4)
  • Chicken liver β‐N‐acetylhexosaminidase (CLH)
  • Additional reagents and equipment for HPLC (see protocol 6) and arrays of enzymatic digests (see protocol 8)
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Figures

Videos

Literature Cited

   Ashford, D., Dwek, R.A., Welply, J.K., Amatayukul, S., Homans, S.W., Lis, H., Taylor, G.N., Sharon, N., and Rademacher, T.W. 1987. The β1‐2‐D‐xylose and α1‐3‐L‐fucose substituted‐N‐linked oligosaccharides from Erythrina crystagalli lectin. Isolation, characterisation and comparison with other legume lectins. Eur. J. Biochem. 166:311‐320.
   Butler, M., Quelhas, D., Critchley, A.J., Carchon, H., Hebestreit, H.F., Hibbert, R.G., Vilarinho, L., Teles, E., Matthijs, G., Schollen, E., Argibay, P., Harvey, D.J., Dwek, R.A., Jaeken, J., and Rudd, P.M. 2003. Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis. Glycobiology 13:601‐622.
   Clausen, H., and Bennett, E.P. 1996. A family of UDP‐GalNAc:polypeptide N‐acetylgalactosaminyl‐transferases control the initiation of mucin‐type O‐linked glycosylation. Glycobiology 6:635‐646.
   Dwek, R.A., Edge, C.J., Harvey, D.A., Wormald, M.W., and Parekh, R.B. 1993. Analysis of glycoprotein associated oligosaccharides. Annu. Rev. Biochem. 62:65‐100.
   Goodarzi, M.T., and Turner, G.A. 1998. Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease. Glycoconj. J. 15:469‐475.
   Guile, G.R., Wong, S.Y., and Dwek, R.A. 1994. Analytical and preparative separation of anionic oligosaccharides by weak anion‐exchange high‐performance liquid chromatography on an inert polymer column. Anal. Biochem. 222:231‐235.
   Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., and Dwek, R.A. 1996. A rapid high‐resolution high‐performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240:210‐226.
   Guile, R.R., Harvey, D.J., O'Donnell, N., Powell, A.K., Hunter, A.P., Zamze, S., Fernandes, D.L., Dwek, R.A., and Wing, D.R. 1998. Identification of highly fucosylated N‐linked oligosaccharides from the human parotid gland. Eur. J. Biochem. 258:623‐656.
   Joao, H.C., Scragg, I.G., and Dwek, R.A. 1992. Effect of glycosylation on protein conformation and amide proton exchange rates in RNase B. FEBS Lett. 307:343‐346.
   Kieffer, B., Driscoll, P.C., Campbell, I.D., Willis, A.C., van der Merwe, P.A., and Davis, S.J. 1994. Three‐dimensional solution structure of the extracellular region of the complement regulatory protein CD59, a new cell‐surface protein domain related to snake venom neurotoxins. Biochem. 33:4471‐4482.
   Ko, K., Tekoah, Y., Rudd, P.M., Harvey, D.J., Dwek, R.A., Spitsin, S., Hanlon, C.A., Rupprecht, C., Dietzschold, B., Golovkin, M., and Koprowski, H. 2003. Function and glycosylation of plant‐derived antiviral monoclonal antibody. Proc. Natl. Acad. Sci. U.S.A. 100:8013‐8018.
   Kuhn, P., Tarentino, A.L., Plummer, T.H. Jr., and Van Roey, P. 1994. Crystal structure of peptide‐N4‐(N‐acetyl‐β‐D‐glucosaminyl)asparagine amidase F at 2.2‐A resolution. Biochemistry. 33:11699‐11706.
   Küster, B., Wheeler, S.F., Hunter, A.P., Dwek, R.A., and Harvey, D.J. 1997. Sequencing of N‐linked oligosaccharides directly from protein gels: In‐gel deglycosylation followed by matrix‐assisted laser desorption/ionization mass spectrometry and normal‐phase high‐performance liquid chromatography. Anal. Biochem. 250:82‐101.
   Kuttner‐Kondo, L., Medof, M.E., Brodbeck, W., and Shoham, M. 1996. Molecular modeling and mechanism of action of human decay‐accelerating factor. Prot. Eng. 9:1143‐1149.
   Mehta, A., Lu, X., Block, T.M., Blumberg, B.S., and Dwek, R.A. 1997. Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: Evidence that a single N‐linked glycosylation site on an HBV envelope protein is crucial for virus secretion—A potential therapeutic target. Proc. Natl. Acad. Sci. U.S.A. 94:1822‐1827.
   Merry, A.H., Neville, D.C, Royle, L., Matthews, B., Harvey, D.J., Dwek, R.A., and Rudd, P.M. 2002. Recovery of intact 2‐aminobenzamide‐labeled O‐glycans released from glycoproteins by hydrazinolysis. Anal. Biochem. 304:91‐99.
   Merry, A.H., Gilbert, R.J., Shore, D.A., Royle, L., Miroshnychenko, O., Vuong, M., Wormald, M.R., Harvey, D.J., Dwek, R.A., Classon, B.J., Rudd, P.M., and Davis, S.J. 2003. O‐glycan sialylation and the structure of the stalk‐like region of the T cell co‐receptor CD8. J. Biol. Chem. 278:27119‐128.
   Norris, G.E., Stillman, T.J., Anderson, B.F., and Baker, E.N. 1994. The three‐dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum. Structure. 2:1049‐1059.
   Peracaula, R., Tabares, G., Royle, L., Harvey, D.J., Dwek, R.A., Rudd, P.M., and de Llorens, R. 2003. Altered glycosylation pattern allows the distinction between prostate‐specific antigen (PSA) from normal and tumor origins. Glycobiology. 13:457‐470.
   Petrescu, A.J., Petrescu, S.M., Dwek, R.A., and Wormald, M.R. 1999. A statistical analysis of N‐ and O‐glycan linkage conformations from crystallographic data. Glycobiology. 9:343‐352.
   Royle, L., Mattu, T.S., Hart, E., Langridge, J.I., Merry, A.H., Murphy, N., Harvey, D.J., Dwek, R.A., and Rudd, P.M. 2002. An analytical and structural database provides a strategy for sequencing O‐glycans from microgram quantities of glycoproteins. Anal. Biochem. 304:70‐90.
   Royle, L., Roos, A., Harvey, D.J., Wormald, M.R., van Gijlswijk‐Janssen, D., Redwan, el‐R.M., Wilson, I.A., Daha, M.R., Dwek, R.A., and Rudd, P.M. 2003. Secretory IgA N‐ and O‐glycans provide a link between the innate and adaptive immune systems. J. Biol. Chem. 278:20140‐20153.
   Rudd, P.M. and Dwek, R.A. 1997a. Glycosylation: Heterogeneity and the 3D structure of proteins. Crit. Rev. Biochem. Mol. Biol. 32:1‐100.
   Rudd, P.M. and Dwek, R.A. 1997b. Rapid, sensitive sequencing of oligosaccharides from glycoproteins. Curr. Opin. Biotechnol. 8:488‐497.
   Rudd, P.M., Guile, G.R., Küster, B., Harvey, D.J., Opdenakker, G., and Dwek, R.A. 1997a. Oligosaccharide sequencing technology. Nature. 388:205‐207.
   Rudd, P.M., Morgan, B.P., Wormald, M.R., Harvey, D.J., van den Berg, C.W., Davis, S.J., Ferguson, M.A.J., and Dwek, R.A. 1997b. The glycosylation of the complement regulatory protein, CD59, derived from human erythrocytes and human platelets. J. Biol Chem. 272:7229‐7244.
   Rudd, P.M., Mattu, T.S., Masure, S., Bratt, T., Van den Steen, P., Wormald, M.R., Küster, B., Harvey, D.J., Borregaard, N., Van Damme, J., Dwek, R.A., and Opdenakker, G. 1999a. Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B‐associated lipocalin. Biochemistry. 38:13937‐13950.
   Rudd, P.M., Endo, T., Colominas, C., Groth, D., Wheeler, S.F., Harvey, D.J., Wormald, M.R., Serban, H., Prusiner, S.B., Kobata, A., and Dwek, R.A. 1999b. Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl. Acad. Sci. U.S.A. 96:13044‐13049.
   Rudd, P.M., Colominas, C., Royle, L., Murphy, N., Hart, E., Merry, A.H., Hebestreit, H.F., and Dwek, R. 2001. A high‐performance liquid chromatography–based strategy for rapid, sensitive sequencing of N‐linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands. Proteomics 1:285‐294.
   Rudd, P.M., Merry, A.H., Wormald, M.R., and Dwek, R.A. 2002. Glycosylation and prion protein. Curr. Opin. Struct. Biol. 12:578‐586.
   Scanlan, C.N., Pantophlet, R., Wormald, M.R., Saphire, E.O., Calarese, D., Stanfield, R., Wilson, I.A., Katinger, H., Dwek, R.A., Burton, D.R., and Rudd, P.M. 2003. The carbohydrate epitope of the neutralizing anti‐HIV‐1 antibody 2G12. Adv. Exp. Med. Biol. 535:205‐218.
   Tekoah, Y., Ko, K., Koprowski, H., Harvey, D.J., Wormald, M.R., Dwek, R.A., and Rudd, P.M. 2004. Controlled glycosylation of therapeutic antibodies in plants. Arch. Biochem. Biophys. 426:266‐278.
   Varki, A. 1993. Biological roles of oligosaccharides: All of the theories are correct. Glycobiology. 3:97‐130.
   Williams, R.L., Greene, S.M., and McPherson, A. 1987. The crystal structure of ribonuclease B at 2.5‐A resolution. J. Biol. Chem. 262:16020‐16031.
   Woods, R.J. 1995. Three dimensional structures of oligosaccharides. Curr. Opin. Struct. Biol. 5:591‐598.
Key References
   Dwek et al., 1993. See above.
  Although some of the technology in this review has been superseded, the chapter remains essential background reading for an understanding of the new technologies. It describes specific analytical techniques required for oligosaccharide analysis, with emphasis on those required for the elucidation of oligosaccharide primary structure.
   Guile et al., 1994. See above.
  This paper describes the methodology for weak anion‐exchange chromatography.
   Guile et al., 1996. See above.
  This study describes a sensitive technology capable of resolving subpicomolar quantities of mixtures containing both neutral and charged fluorescently labeled oligosaccharides in a single run using normal‐phase HPLC. It describes how incremental values for the addition of monosaccharides to oligosaccharide cores have been calculated, enabling the prediction of structures from the elution positions of oligosaccharides.
   Petrescu et al., 1999. See above.
  A recently compiled database that allows a more full understanding of the implications of N‐ and O‐glycosylation for the structure and function of a protein, which can only be reached when a glycoprotein is viewed as a single entity.
   Royle et al., 2002. See above.
  This study describes the strategy and techniques for the sequencing of O‐glycans from micrograms of glycoproteins.
   Rudd et al., 1999b. See above.
  A paper showing the N‐glycan analysis of a glycan pool containing 52 glycans with different compositions. It also shows how to use enzyme digestion profiles to make comparisons of glycosylation.
Internet Resources
   http://www.cbs.dtu.dk/databases/OGLYCBASE
  Site for O‐GlycBase, a database of O‐glycosylated proteins, and for O‐Unique, a database of O‐glycosylated mucins.
   http://bssv01.lancs.ac.uk/gig/pages/gag/carbbank.htm
  Site for the complex carbohydrate structure database (CCSD16), which can be used to perform searches for carbohydrate structures. Additionally, SUGABASE is a carbohydrate‐NMR database that combines CCSD with proton and carbon chemical shift values.
   http://www.ccrc.uga.edu
  The site for the Complex Carbohydrate Research Centre Neural Networks (CCRC‐Net) contains mass spectra of partially methylated alditol acetate derivatives generated during carbohydrate methylation analysis.
   http://us.expasy.org/tools/glycomod/
  A tool for predicting oligosaccharide structures on proteins from experimentally determined masses. Can be used for free or derivatized oligosaccharides or glycopeptides.
   http://www.dkfz‐heidelberg.de/spec/sweet2/doc/index.html
  A program for constructing three‐dimensional models of saccharides from their sequences.
   http://www.bioch.ox.ac.uk/glycob/
  Web site for the Oxford Glycobiology Institute.
  http://www.eurocarbdb.org/
  The EUROCarbDB design study aims to create the foundations for databases and bioinformatics tools in the realm of glycobiology and glycomics, and will establish mainly the technical framework for bottom‐to‐top initiatives where all interested research groups can enter their primary data. The new infrastructure will constitute the nucleus for the creation of a depository for carbohydrate‐related data similar to the extensively used data collections in the areas of genomics and proteomics.
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