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Mol Cell Proteomics. 2012 Jun;11(6):M111.015339. doi: 10.1074/mcp.M111.015339. Epub 2012 Jan 19.

Mapping yeast N-glycosites with isotopically recoded glycans.

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  • 1Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.

Abstract

Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.

PMID:
22261724
[PubMed - indexed for MEDLINE]
PMCID:
PMC3433913
Free PMC Article

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