Analysis of Oligosaccharide Negative Charge by Anion‐Exchange Chromatography

Ajit Varki1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.20
DOI:  10.1002/0471142727.mb1720s32
Online Posting Date:  May, 2001
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Abstract

This unit presents the analysis of negative charge on labeled N‐ or O‐linked oligosaccharides. These protocols may be used in the initial screening of oligosaccharides to detect negative charge, for analytical or preparative separation of oligosaccharides based on their negative charge, or to analyze the type of negative charge found on the oligos accharides. The describes the use of anion‐exchange (QAE‐Sephadex) chromatography with stepwise elution for estimating the number of negative charges on an oligosaccharide sample derived from glycosidase treatment of a glycoprotein. In an , gradient elution is used for the preparative separation of oligosaccharides based on negative charge. A describes a method for measuring loss of or change in negative charge after treatment of the oligosaccharide sample with mild acid and/or phosphatases.

     
 
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Table of Contents

  • Basic Protocol 1: Separation and Analysis of Anionic Oligosaccharices by QAE‐Sephadex Chromatography with Stepwise Elution
  • Alternate Protocol 1: Separation and Analysis of Anionic Oligosaccharides by QAE‐Sephadex Chromatography with Gradient Elution
  • Support Protocol 1: Detection and Removal of Phosphodiesters or Phosphomonoesters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Separation and Analysis of Anionic Oligosaccharices by QAE‐Sephadex Chromatography with Stepwise Elution

  Materials
  • Radiolabeled mixture of oligosaccharides released from glycoprotein (units 17.12 17.17)
  • recipeEquilibrated QAE‐Sephadex chromatography resin (see recipe)
  • 2 mM Tris base
  • recipeElution buffers (see recipe)
  • 1‐ to 2‐ml Pasteur pipets plugged with glass wool or 1‐ to 2‐ml disposable plastic columns
  • Sintered‐glass funnel
  • Additional reagents and equipment for metabolic radiolabeling (unit 17.4) and autoradiography ( appendix 3A)

Alternate Protocol 1: Separation and Analysis of Anionic Oligosaccharides by QAE‐Sephadex Chromatography with Gradient Elution

  • 5‐ to 20‐ml disposable plastic column
  • Gradient mixer
  • Additional materials and equipment for salt gradient preparation (unit 10.10)

Support Protocol 1: Detection and Removal of Phosphodiesters or Phosphomonoesters

  • Radiolabeled mixture of oligosaccharides released from glycoprotein (units 17.12 17.17), desalted and lyophilized
  • 10 U/ml E. coli alkaline phosphatase
  • 2 M acetic acid
  • 200 mM Tris⋅Cl, pH 8.0 ( appendix 22)
  • Strong 10‐ml conical glass tubes or 1‐ml Reacti‐Vials (Pierce)
  • Water bath or heating block 80°C
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Figures

Videos

Literature Cited

   Cummings, R.D., Merkle, R.K., and Stults, N.L. 1989. Separation and analysis of glycoprotein oligosaccharides. Methods Cell. Biol. 32:141‐183.
   Fukuda, M. 1989. Characterization of O‐linked saccharides from cell surface glycoproteins. Methods Enzymol. 179:17‐29.
   Goldberg, D.E. and Kornfeld, S. 1981. The phosphorylation of β‐glucuronidase oligosaccharides in mouse P388D1 cells. J. Biol. Chem. 256:13060‐13067.
   Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. 1988. Sulfated N‐linked oligosaccharides in mammalian cells I: Complex‐type chains with sialic acids and O‐sulfate esters. J. Biol. Chem. 236:8879‐8889.
   Tabas, I. and Kornfeld, S. 1980. Biosynthetic intermediates of β‐glucuronidase contain high mannose oligosaccharides with blocked phosphate residues. J. Biol. Chem. 255:6633‐6639.
   van Pelt, J., Damm, J.B., Kamerling, J.P., and Vliegenthart, J.F.G. 1987. Separation of sialyl‐oligosaccharides by medium pressure anion‐exchange chromatography on Mono Q. Carbohydr. Res. 169:43‐51.
   Varki, A. and Kornfeld, S. 1980. Structural studies of phosphorylated high mannose–type oligosaccharides. J. Biol. Chem. 255:10847‐10858.
   Varki, A. and Kornfeld, S. 1983. The spectrum of anionic oligosaccharides released by endo‐β‐N‐acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor. J. Biol. Chem. 258:2808‐2818.
Key Reference
   Varki, A. and Kornfeld, S. 1980. See above.
  Figures in the miniprint section of this paper provide several examples of the types of analyses described here.
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