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Updated: Jul 5, 2026
10:17
Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
Published on: April 27, 2010
Release of saccharides from glycoconjugates
L D Powell1, A P Varki, H H Freeze
1University of California San Diego, La Jolla, California, USA.
Current Protocols in Immunology
|April 25, 2008
Summary
Glycosidases, enzymes that remove sugar chains, are detailed for analyzing cell-surface glycoconjugates. Protocols cover exoglycosidases, endoglycosidases, and glycoamidases for glycoprotein and glycopeptide analysis.
Area of Science:
- Biochemistry and enzymology
- Glycobiology and glycoconjugate analysis
Background:
- Glycosidases are crucial enzymes for modifying and analyzing carbohydrate structures on cell surfaces.
- Understanding glycosidase function is key to characterizing glycoproteins and glycopeptides.
Purpose of the Study:
- To provide detailed protocols for the use of various glycosidases in biochemical analysis.
- To enable researchers to characterize N-linked carbohydrate chains on proteins and glycopeptides.
Main Methods:
- Detailed protocols for exoglycosidase, endoglycosidase, and glycoamidase digestion.
- Sialidase digestion of purified proteins and intact cell suspensions.
- Enzyme activity testing and sequential digestion strategies for carbohydrate chain analysis.
Main Results:
- Established protocols for common endoglycosidases (Endo H, Endo F2, PNGase F) and glycoamidases.
- Demonstrated methods for estimating the number and types of N-linked carbohydrate chains.
- Described techniques for recovering and analyzing released radiolabeled oligosaccharide chains.
Conclusions:
- The provided protocols offer a comprehensive toolkit for glycosidase-based analysis of glycoconjugates.
- These methods facilitate detailed structural and quantitative characterization of N-linked glycans.
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