Detection of Individual Glycosylation Sites on Glycoproteins

Leland D. Powell1

1 University of California San Diego, School of Medicine, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.14B
DOI:  10.1002/0471142727.mb1714bs32
Online Posting Date:  May, 2001
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Abstract

In this unit, glycopeptides generated by endopeptidase digestion are first separated by reversed‐phase chromatography. The presence of hydrophilic, negatively charged oligosaccharides shortens retention times, causing glycopeptides to elute in considerably broader peaks than do peptides, so by following the elution profile either radiochemically or colorimetrically, the peaks corresponding to unique glycopeptides can be identified. With proper controls, the number of peaks will correspond to the number of different glycosylation sites. The eluted fractions are suitable for analysis by lectin chromatography, and the peptide sugar linkage can be defined either by endoglycosidase digestion or chemical cleavage. Oligosaccharides freed from the peptide according to the methods described in this unit can be characterized by size or charge, techniques not generally applicable with glycopeptides.

     
 
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Table of Contents

  • Basic Protocol 1: Fractionation of Glycopeptides by Reversed‐Phase HPLC
  • Support Protocol 1: Endoglycosidase Digestion of Purified Glycopeptides
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Fractionation of Glycopeptides by Reversed‐Phase HPLC

  Materials
  • Highly purified glycoprotein metabolically labeled with a radioactive sugar precursor (unit 17.4)
  • recipeHPLC buffers A and B (see recipe)
  • 1 M Tris⋅Cl, pH 8.4 ( appendix 22)
  • Organic solvents: chloroform, DMSO, acetonitrile, and methanol
  • recipe10 mM ammonium formate in water and in 50% acetonitrile (see recipe)
  • Ventilated oven, 40° to 45°C
  • Nitrogen tank
  • C 18 cartridge (e.g., Sep‐Pak cartridge, Waters)
  • 1‐cc and glass 10‐cc syringes
  • 50‐ml polypropylene tube
  • Additional reagents and equipment for peptide isolation by reversed‐phase HPLC (unit 10.12)

Support Protocol 1: Endoglycosidase Digestion of Purified Glycopeptides

  • Glycopeptide sample (see protocol 1, step )
  • HPLC apparatus with 500‐µl injector sample loop
  • Additional reagents and equipment for endoglycosidase digestion (unit 17.13)
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Figures

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Literature Cited

Literature Cited
   Dahms, N.M. and Hart, G.W. 1985. Lymphocyte function‐associated antigen 1 contains sulfated N‐linked oligosaccharides. J. Immunol. 134:3978‐3986.
   Kobata, A. and Takasaki, S. 1992. Structure and biosynthesis of cell surface carbohydrates. In Cell Surface Carbohydrates and Development (M. Fukuda, ed.) pp. 1‐24.
   Swiedler, S.J., Hart, G.W., Tarentino, A.L., Plummer, T.H., and Freed, J.H. 1983. Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B‐cell lymphoma. J. Biol. Chem. 258:11515‐11523.
   Treuheit, M.J., Costello, C.E., and Halsall, H.B. 1992. Analysis of the five glycosylation sites of human α1‐acid glycoprotein. Biochem. J. 283:105‐112.
Key Reference
   Swiedler et al., 1983. See above.
  The reference upon which most of these methods are based.
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