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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M900211-MCP200v1 8/11/2515    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Google Scholar Articles by Darula, Z. Articles by Medzihradszky, K. F. PubMed PubMed Citation Articles by Darula, Z. Articles by Medzihradszky, K. F. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:2515-2526, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Affinity Enrichment and Characterization of Mucin Core-1 Type Glycopeptides from Bovine Serum^*,

Zsuzsanna Darula and Katalin F. Medzihradszky^,,¶

From the Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences , Szeged P. O. Box 521, Szeged H-6701, Hungary and
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158-2517

The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Galβ1–3GalNAc (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > 850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis.

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