Preparation of Glycopeptides

Leland D. Powell1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.14A
DOI:  10.1002/0471142727.mb1714as32
Online Posting Date:  May, 2001
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Abstract

Generation of glycopeptides from glycoproteins is frequently useful when analyzing a protein's oligosaccharide side chains. Freed from the bulk of the polypeptide backbone by proteolysis, glycopeptides can be characterized by a variety of techniques. Extensive proteolysis with pronase or proteinase K results in oligosaccharides with one or a few amino acid residues attached. This technique, detailed in this unit, is often employed as a first step in characterizing oligosaccharides on very large glycoproteins such as proteoglycans and mucins. Limited proteolysis with a specific endoproteinase (e.g., trypsin, a‐chymotrypsin, and V8 protease) is also described, and leaves a larger peptide attached to the oligosaccharide. The resulting glycopeptides are generally suitable substrates for Peptide:N‐glycosidase F, an enzyme useful in defining oligosaccharide‐peptide linkages. Additionally, they can be separated by reversed‐phase chromatography, resulting in a glycopeptide map that is analogous to a peptide map, and used for detection of glycosylation sites.

     
 
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Table of Contents

  • Basic Protocol 1: Extensive Proteolytic Digestion of Glycoproteins
  • Basic Protocol 2: Proteolytic Digestion of Purified Glycoproteins with Endopeptidases
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Extensive Proteolytic Digestion of Glycoproteins

  Materials
  • Radiolabeled glycoprotein sample (unit 17.4)
  • 20% (w/v) sodium dodecyl sulfate (SDS)
  • 1 M 2‐mercaptoethanol (2‐ME)
  • Sephacryl S‐200 gel‐filtration column (50‐ to 75‐ml bed volume; Sigma or Pharmacia Biotech), equilibrated (unit 10.9) in recipeammonium formate/azide solution containing 0.1% (w/v) SDS (see recipe)
  • 100% and 85% (v/v) acetone (HPLC or ACS grade), ice‐cold
  • 0.1 M Tris⋅Cl, pH 7.5 ( appendix 22), without or with 10 mM CaCl 2 (Tris/CaCl 2)
  • Pronase or proteinase K stock solution
  • Phenylmethylsulfonylfluoride (PMSF), in 100% ethanol
  • Sephadex G‐15 (Sigma or Pharmacia Biotech) or Bio‐Gel P‐2 (Bio‐Rad) columns (30‐ to 60‐ml bed volume), equilibrated in recipeammonium formate/azide solution
  • 15‐ or 50‐ml conical polyproplyene tubes
  • Beckman TJ‐6 centrifuge or equivalent
  • Water bath, 50°C
  • Additional reagents and equipment for preparation and standardization of gel‐filtration columns and sample desalting (unit 10.9), quantitation of proteins (unit 10.110.1A), and acetone precipitation (unit 17.1017.10A)

Basic Protocol 2: Proteolytic Digestion of Purified Glycoproteins with Endopeptidases

  Materials
  • Radiolabeled protein sample (unit 17.4)
  • Tris/SDS solution
  • Dithiothreitol (DTT)
  • Iodoacetamide
  • 1N sodium hydroxide (NaOH), prepared fresh every 4 weeks
  • 2‐mercaptoethanol (2‐ME), undiluted
  • Sephadex G‐50 column (30‐ to 60‐ml bed volume; Sigma or Pharmacia Biotech) column, equilibrated in ammonium formate/azide solution containing 0.1% (w/v) SDS (unit 10.9)
  • 100% and 85% (v/v) acetone, ice‐cold
  • Nitrogen (N 2) stream
  • recipeProtease digestion buffer
  • recipeProtease stock solution
  • 5‐ to 15‐ml Pyrex or polypropylene tubes
  • Aluminum foil
  • pH paper, range pH 7 to 12
  • 10‐ml round‐bottomed polypropylene tubes
  • Beckman TJ‐6 centrifuge or equivalent
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Additional reagents and equipment for preparation and standardization of gel‐filtration columns and sample desalting (unit 10.9) and acetone precipitation (unit 17.1017.10A)
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Figures

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Literature Cited

Literature Cited
   Britz, J.S. and Hart, G.W. 1983. Biosynthesis of glycosaminoglycans by epithelial and lymphocytic components of murine thymus. J. Immunol. 130:1848‐1855.
   Drapeau, G.R. 1977. Cleavage at glutamic acid with Staphylococcal protease. Methods Enzymol. 47:189‐191.
   Ebeling, W., Hennrich, N., Clockow, M., Metz, H., Orth, H.D., and Lang, H. 1974. Proteinase K from Tritirachium album Limber. Eur. J. Biochem. 47:91‐97.
   Finne, J. and T., Krusius 1982. Preparation and fractionation of glycopeptides. Methods Enzymol. 83:269‐277.
   Judd, R.C. 1990. Peptide mapping. Methods Enzymol. 182:613‐626.
   Spiro, R.G. 1966. Characterization of carbohydrate units of glycoproteins. Methods Enzymol. 8:26‐52.
   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.
   Takasaki, S., Mizuochi, T., and Kobata, A. 1982. Hydrazinolysis of asparagine‐linked sugar chains to produce free oligosaccharides. Methods Enzymol. 83:263‐268.
Key References
   Judd, 1990. See above.
  Useful review on specific endopeptidases.
   Spiro, 1966. See above.
  Describes use of pronase and techniques for handling glycopeptides.
   Swiedler et al., 1983. See above.
  Technique from which the basic protocol on endoproteinase digestion was adapted.
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