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

https://doi.org/10.1074/mcp.M900211-MCP200Get rights and content
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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 Galβ1–3GalNAcα (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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*

This work was supported, in whole or in part, by National Institutes of Health Grants RR001614 and RR019934 from the National Center for Research Resources (to the University of California San Francisco Mass Spectrometry Facility; director, A. L. Burlingame). This work was also supported by Hungarian Science Foundation Grant OTKA T60283 (to K. F. M.).

The on-line version of this article (available at http://www.mcponline.org) contains supplemental Figs. 1–32 and Tables 1 and 2.