UNIT 17.14A Preparation of Glycopeptides
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Powell, L. D. 2001. Preparation of Glycopeptides. Current Protocols in Molecular Biology. 32:III:17.14A:17.14.1–17.14.9.
- Published Online: 1 MAY 2001
- Published Print: OCT 1995
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.