Determining the Structure of Glycan Moieties by Mass Spectrometry

David J. Harvey1, Raymond A. Dwek1, Pauline M. Rudd1

1 University of Oxford, Oxford
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.7
DOI:  10.1002/0471140864.ps1207s43
Online Posting Date:  March, 2006
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Abstract

Acquisition of mass spectra using fast‐atom bombardment (FAB), matrix‐assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) mass spectrometry is summarized in this unit. Molecular weights of the carbohydrates provide information on their composition in terms of isobaric monosaccharide composition, and tables of residue masses are provided to assist these calculations. More detailed structural analysis can be performed by fragmentation; the main fragmentation modes of carbohydrates and their nomenclature are discussed. Fragmentation of negative ions generated by nano‐electrospray, in particular, provide very informative spectra and give details such as the position of fucose substitution, branching pattern, and the presence or absence of bisecting GlcNAc residues. Practical details are given for the purification of samples prior to mass spectrometric analysis and for recording FAB, MALDI, and ESI spectra.

     
 
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Table of Contents

  • Calculation of Mass
  • Fragmentation
  • Sample Purity
  • Basic Protocol 1: Analysis of Glycans by Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry
  • Basic Protocol 2: Analysis of Glycans by Fast Atom Bombardment Mass Spectrometry
  • Basic Protocol 3: Analysis of Glycans by Electrospray Ionization Mass Spectrometry
  • Support Protocol 1: Removal of Contaminants by Microdialysis or Adsorption onto Membranes
  • Support Protocol 2: Purification of N‐Linked Oligosaccharides on a Porous Graphitized Carbon Column
  • Support Protocol 3: Purification of 2‐AB Derivatives of Neutral N‐Linked Glycans Using a C18 ZipTip
  • Support Protocol 4: Purification of Neutral Glycans Released from SDS Gels
  • Support Protocol 5: Purification of Sialylated Glycans Released from SDS Gels
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Glycans by Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry

  Materials
  • Glycan sample, purified (see Support Protocols protocol 41 to protocol 85 and unit 12.6)
  • Matrix solution (saturated solution of DHB in acetonitrile; see recipe)
  • Mass spectrometer (e.g., Waters Micromass MALDI Micro, Applied Biosystems Voyager series TOF mass spectrometer, Bruker “flex” series MALDI‐TOF mass spectrometer, or Shimadzu Kratos Axima CFR)

Basic Protocol 2: Analysis of Glycans by Fast Atom Bombardment Mass Spectrometry

  Materials
  • Sodium hydroxide (NaOH) pellets
  • Dry dimethyl sulfoxide (DMSO)
  • Purified glycan sample (see Support Protocols protocol 41 to protocol 85 and unit 12.6)
  • Methyl iodide
  • Chloroform
  • Source of dry nitrogen
  • 50% (v/v) methanol/water
  • 75% (v/v) acetonitrile/water
  • 1:1 (v/v) glycerol/thioglycerol
  • Mass spectrometer (e.g., Micromass AutoSpec or Finnigan ThermoQuest 900)
  • Additional reagents and equipment for cleaning sample (see Support Protocols protocol 41, protocol 63, and protocol 74)

Basic Protocol 3: Analysis of Glycans by Electrospray Ionization Mass Spectrometry

  Materials
  • Sample prepared by PNGase F digestion or hydrazinolysis (unit 12.6)
  • Membrane (select one):
  •  Dialysis membrane (e.g., Spectra/Pore CE membrane, MWCO 500)
  •  Nafion 117 membrane (Aldrich)

Support Protocol 1: Removal of Contaminants by Microdialysis or Adsorption onto Membranes

  Materials
  • PNGase F digestion sample (unit 12.6)
  • 1 M NaOH
  • 25% (v/v) aqueous acetonitrile
  • 25% (v/v) aqueous acetonitrile/0.05% (v/v) trifluoroacetic acid
  • 80% (v/v) aqueous acetonitrile/ 0.10% (v/v) trifluoroacetic acid
  • 100‐mg prepacked Hypersep porous graphitized carbon (PGC) columns (ThermoQuest)

Support Protocol 2: Purification of N‐Linked Oligosaccharides on a Porous Graphitized Carbon Column

  Materials
  • Sample prepared by PNGase F digestion or hydrazinolysis and derivatized by reductive amination (unit 12.6)
  • C18 ZipTips (Millipore)
  • 1:1 (v/v) acetonitrile/water

Support Protocol 3: Purification of 2‐AB Derivatives of Neutral N‐Linked Glycans Using a C18 ZipTip

  Materials
  • Sample prepared by in‐gel PNGase F release (unit 12.6)
  • Conditioned AG50X12 resin (see recipe)
  • Conditioned AG3X4 resin (see recipe)
  • C18 resin: obtain from a SepPak cartridge and make a slurry in ethanol
  • Pressurized nitrogen
  • GelLoader pipet tip (Eppendorf)
  • SpeedVac evaporator

Support Protocol 4: Purification of Neutral Glycans Released from SDS Gels

  • Sample of sialylated glycans prepared by in‐gel PNGase F release (unit 12.6)
  • AG50X12 resin (wash 100 µl resin with 1 ml of 1 M NaOH solution followed by an equivalent amount of water)
  • Anhydrous dimethyl sulfoxide (DMSO), stored over a molecular sieve
  • Methyl iodide
  • 20% (v/v) acetonitrile in water
  • GelLoader pipet tips (Eppendorf)
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Figures

Videos

Literature Cited

   Börnsen, K.O., Mohr, M.D., and Widmer, H.M. 1995. Ion exchange and purification of carbohydrates on a Nafion membrane as a new sample pretreatment for matrix‐assisted laser desorption‐ionization mass spectrometry. Rapid Commun. Mass Spectrom. 9:1031‐1034.
   Charlwood, J., Langridge, J., Tolson, D., Birrell, H., and Camilleri, P. 1999. Profiling of 2‐aminoacridone derivatised glycans by electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 13:107‐112.
   Chen, P., Baker, A.G., and Novotny, M.V. 1997. The use of osazones as matrices for the matrix‐assisted laser desorption/ionization mass spectrometry of carbohydrates. Anal. Biochem. 244:144‐151.
   Ciucanu, I. and Kerek, F. 1984. A simple and rapid method for the permethylation of carbohydrates. Carbohydrate Res. 131:209‐217.
   Domon, B. and Costello, C.E. 1988. A systematic nomenclature for carbohydrate fragmentations in FAB‐MS/MS spectra of glycoconjugates. Glycoconjugate J. 5:397‐409.
   Harvey, D.J., Bateman, R.H., and Green, M.R. 1997. High energy collision‐induced fragmentation of complex oligosaccharides ionised by matrix‐assisted laser desorption mass spectrometry. J. Mass Spectrom. 32:167‐187.
   Harvey, D.J. 1993. Quantitative aspects of the matrix‐assisted laser desorption mass spectrometry of complex oligosaccharides. Rapid Commun. Mass Spectrom. 7:614‐619.
   Harvey, D.J. 1999. Matrix‐assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom. Rev. 18:349‐451.
   Harvey, D.J. 2000. Collision‐induced fragmentation of underivatized N‐linked carbohydrates ionized by electrospray. J. Mass Spectrom. 35:1178‐1190.
   Harvey, D.J. 2005a. Fragmentation of negative ions from carbohydrates: Part 1, Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N‐linked carbohydrates. J. Am. Soc. Mass Spectrom. 16:622‐630.
   Harvey, D.J. 2005b. Fragmentation of negative ions from carbohydrates: Part 2, Fragmentation of high‐mannose N‐linked glycans. J. Am. Soc. Mass Spectrom. 16:631‐646.
   Harvey, D.J. 2005c. Fragmentation of negative ions from carbohydrates: Part 3, Fragmentation of hybrid and complex N‐linked glycans. J. Am. Soc. Mass Spectrom. 16:647‐659.
   Karas, M., Ehring, H., Nordhoff, E., Stahl, B., Strupat, K., Hillenkamp, F., Grehl, M., and Krebs, B. 1993. Matrix‐assisted laser desorption/ionization mass spectrometry with additives to 2,5‐dihydrobenzoic acid. Org. Mass Spectrom. 28:1476‐1481.
   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.
   Mechref, Y. and Novotny, M.V. 1998. Matrix‐assisted laser desorption/ionization mass spectrometry of acidic glycoconjugates facilitated by the use of spermine as a co‐matrix. J. Am. Soc. Mass Spectrom. 9:1292‐1302.
   Metzger, J.O., Woisch, R., Tuszynski, W., and Angermann, R. 1994. New type of matrix for matrix‐assisted laser desorption mass spectrometry of polysaccharides and proteins. Fresenius J. Anal. Chem. 349:473‐474.
   Mo, W., Sakamoto, H., Nishikawa, A., Kagi, N., Langridge, J.I., Shimonishi, Y., and Takao, T. 1999. Structural characterisation of chemically derivatised oligosaccharides by nanoflow electrospray ionization mass spectrometry. Anal. Chem. 71:4100‐4106.
   Mock, K.K., Sutton, C.W., and Cottrell, J.S. 1992. Sample immobilization protocols for matrix‐assisted laser‐desorption mass spectrometry. Rapid Commun. Mass Spectrom. 6:2333‐238.
   Mohr, M.D., Börnsen, K.O., and Widmer, H.M. 1995. Matrix‐assisted laser desorption/ionization mass spectrometry: Improved matrix for oligosaccharides. Rapid Commun. Mass Spectrom. 9:809‐814.
   Packer, N.H., Lawson, M.A., Jardine, D.R., and Redmond, J.W. 1998. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconjugate J. 15:737‐747.
   Pitt, J.J. and Gormon, J.J. 1996. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry of sialylated glycopeptides and proteins using 2,6‐dihydroxyacetophenone as a matrix. Rapid Commun. Mass Spectrom. 10:1786‐1788.
   Powell, A.K. and Harvey, D.J. 1996. Stabilisation of sialic acids in N‐linked oligosaccharides and gangliosides for analysis by positive ion matrix‐assisted laser desorption‐ionization mass spectrometry. Rapid Commun. Mass Spectrom. 10:1027‐1032.
Key References
   Dwek, R.A., Edge, C.J., Harvey, D.J., Wormald, M.W., and Parekh, R.B. 1993. Analysis of glycoprotein associated oligosaccharides. Annu. Rev. Biochem. 62:65‐100.
  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.
   Küster et al., 1997. See above.
  This paper describes a sensitive technique for releasing N‐linked glycans from within SDS‐PAGE gels and for their analysis by MALDI mass spectrometry.
   Harvey, D.J. 1999. Matrix‐assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom. Rev. 18:349‐451.
  This is a comprehensive review of MALDI mass spectrometry as applied to carbohydrate analysis covering instrumentation, techniques, sample preparation, and applications from the literature to the end of 1998.
Internet Resources
   http://www.cbs.dtu.dk/services/
  Center for Biological Sequence Analysis site for NetOGlyc and NetNGlyc, which can be used to predict O‐ and N‐glycosylation sites in glycosylated proteins.
   https://tmat.proteomesystems.com/glycosuite
  The GlycoSuite database (GlycoSuiteDB) is an annotated and curated relational database of glycan structures containing most published O‐linked glycans, as well as N‐linked glycans in the literature from the years 1990‐2003. Information includes glycan type, structure, mass and composition, sources, (tissue and/or cell type etc.), and, where known, the proteins to which the glycans are attached. Cross‐references to SWISS‐PROT/TrEMBL are given if applicable, and literature references are linked to PubMed. Bioinformatics tools for interpretation of mass spectrometric data are also provided, including the following programs (all registered trademarks of Proteome Systems Ltd.): GlycosidIQ (a glycofragment mass fingerprinting tool which takes MS/MS data and predicts the possible structures of the oligosaccharide); GlycoComp (a calculator of glycan compositions corresponding to parent mass data of released glycans or glycopeptides); GlycoBuild (with which the user can construct a 2D oligosaccharide structure); and GlycoFrag (with which the user can theoretically fragment an entered structure to compare with experimental data).
   http://www.dkfz‐heidelberg.de/spec2/sweetdb/
  Contains an extensive, searchable database of carbohydrate structures, a database of publications on carbohydrates linked to PubMed, and a tool for mass spectral interpretation.
   http://www.functionalglycomics.org/static/consortium/links.shtml
  Consortium for Functional Glycomics. Contains links to databases, information on carbohydrate nomenclature, discussion groups, etc.
   http://www.ccrc.uga.edu
  The site for the Complex Carbohydrate Research Center Neural Networks (CCRC‐Net) contains mass spectra of partially methylated alditol acetate derivatives generated during carbohydrate methylation analysis.
   http://afmb.cnrs‐mrs.fr/CAZY/index.html
  Information on carbohydrate‐active enzymes.
   http://www.bioch.ox.ac.uk/glycob/
  Web site for the Oxford Glycobiology Institute.
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