β‐Elimination for Release of O‐GalNAc‐Linked Oligosaccharides from Glycoproteins and Glycopeptides

Minoru Fukuda1

1 La Jolla Cancer Research Foundation, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.15B
DOI:  10.1002/0471142727.mb1715bs31
Online Posting Date:  May, 2001
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Abstract

This unit describes release of oligosaccharides that are attached to polypeptides through an N‐acetylgalactosamine (GalNAc) linkage to the hydroxyl groups of serine or threonine. The b‐elimination procedures described here can be used to recover the oligosaccharide chains (also called glycans) and/or identify the serine or threonine residues involved in the linkage. A b‐elimination method employing sodium borohydride (NaBH4) and alkaline conditions is described, and an alternative method is also presented in which only alkaline conditions are used without a reducing agent. Another alternative protocol uses sodium sulfite.

     
 
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Table of Contents

  • Basic Protocol 1: β‐Elimination in the Presence of Sodium Borohydride
  • Alternate Protocol 1: Rapid β‐Elimination in Dimethyl Sulfoxide Solution
  • Alternate Protocol 2: β‐Elimination in the Presence of Sodium Sulfite
  • Support Protocol 1: Separation of β‐Elimination Products by Paper Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: β‐Elimination in the Presence of Sodium Borohydride

  Materials
  • Glycopeptide or glycoprotein sample
  • 1 M NaBH 4/0.05 M NaOH (prepare fresh)
  • 1 M acetic acid in methanol (prepare fresh, cool to room temperature before use)
  • Nitrogen gas stream
  • Methanol (HPLC grade)
  • 0.2% orcinol/2 M H 2SO 4 (store at 4°C)
  • 9‐ml screw‐cap conical glass test tubes
  • 25° to 30°C and 45°C water baths
  • 1 × 30–cm Sephadex G‐15 column (Pharmacia Biotech) equilibrated in H 2O
  • Silica gel–coated thin‐layer chromatography (TLC) plate
  • 150° to 200°C oven

Alternate Protocol 1: Rapid β‐Elimination in Dimethyl Sulfoxide Solution

  • Dimethyl sulfoxide (DMSO)
  • 0.425 M KOH
  • 100% ethanol
  • 0.4 M HCl

Alternate Protocol 2: β‐Elimination in the Presence of Sodium Sulfite

  • 0.5 M Na 2SO 3/0.1 M NaOH (prepare fresh)
  • 1 M and 6 M HCl
  • 7‐ml conical glass test tube
  • Vacuum evaporator, 40°C
  • 110°C oven

Support Protocol 1: Separation of β‐Elimination Products by Paper Chromatography

  Materials
  • Dried β‐elimination reaction product (see protocol 3)
  • 0.05 M NaOH
  • Dowex AG‐1‐X8 resin (200 to 400 mesh; formate form; Bio‐Rad)
  • recipe5 mM pyridine formate buffer, pH 4.0 (see recipe)
  • 4 M formic acid
  • 4:1:5 (v/v/v) 1‐butanol/acetic acid/water
  • 0.5 × 5–cm chromatography column
  • Whatman 3 MM chromatography paper
  • Paper chromatography chamber
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Figures

Videos

Literature Cited

Literature Cited
   Carlson, D.M. 1968. Structures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins. J. Biol. Chem. 243:616‐626.
   Downs, F., Herp, A., Moschera, J., and Pigman, W. 1973. β‐elimination and reduction reactions and some applications of dimethylsulfoxide on submaxillary glycoproteins. Biochim. Biophys. Acta 328:182‐192.
   Fukuda, M. 1989. Characterization of O‐linked saccharides from cell surface glycoproteins. Methods Enzymol. 179:17‐29.
   Ogata, S.‐I. and Lloyd, K.O. 1982. Mild alkaline borohydride treatment of glycoproteins—A method for liberating both N‐ and O‐linked carbohydrate chains. Anal. Biochem. 119:351‐359.
   Sojar, H.T. and Bahl, O.P. 1987. Chemical deglyosylation of glycoproteins. Methods Enzymol. 138:350‐359.
   Spiro, R.G. 1972. Study of carbohydrates of glycoproteins. Methods Enzymol. 28:3‐43.
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