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Analysis of Oligosaccharide Negative Charge by Anion‐Exchange Chromatography

Ajit Varki¹

¹University 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 Basic Protocol 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 Alternate Protocol, gradient elution is used for the preparative separation of oligosaccharides based on negative charge. A Support Protocol 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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Unit Introduction
Basic Protocol: Separation and Analysis of Anionic Oligosaccharices by QAE-Sephadex Chromatography with Stepwise Elution
Alternate Protocol: Separation and Analysis of Anionic Oligosaccharides by QAE-Sephadex Chromatography with Gradient Elution
Support Protocol: Detection and Removal of Phosphodiesters or Phosphomonoesters
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

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

Materials

Radiolabeled mixture of oligosaccharides released from glycoprotein (UNITS 17.12-17.17A)
Equilibrated QAE-Sephadex chromatography resin (see recipe)
2 mM Tris base
Elution 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: Separation and Analysis of Anionic Oligosaccharides by QAE-Sephadex Chromatography with Gradient Elution

Additional Materials (also see Basic Protocol)

5- to 20-ml disposable plastic column
Gradient mixer

Additional materials and equipment for salt gradient preparation (UNIT 10.10)

Support Protocol: Detection and Removal of Phosphodiesters or Phosphomonoesters

Additional Materials (also see Basic Protocol)

Radiolabeled mixture of oligosaccharides released from glycoprotein (UNITS 17.12-17.17A), desalted and lyophilized
10 U/ml E. coli alkaline phosphatase
2 M acetic acid
200 mM Tris×Cl, pH 8.0 (APPENDIX 2)

Strong 10-ml conical glass tubes or 1-ml Reacti-Vials (Pierce)
Water bath or heating block 80°C

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Figures

Figure 17.20.1

Schematic examples of QAE-Sephadex analysis (based on Varki and Kornfeld, 1983). Numbers in italics indicate the charges on the high mannose–type oligosaccharides eluting at that position. (A) Gradient fractionation of mixture of N-linked oligosaccharides with different combinations of sialic acid phosphomonoesters or phosphodiesters. (B) Peak marked “–1” consists of a mixture of oligosaccharides with one negative charge due to either one sialic acid residue (acid- or sialidase-sensitive) or one phosphodiester (increased negative charge after mild acid, sensitive to alkaline phosphatase only after mild acid treatment). The procedures used to evaluate oligosaccharide mixtures are listed on the right with examples of resulting shifts in the elution peaks for each type of treatment illustrated on the left.

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Literature Cited

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 -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 -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--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.