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Reference: Breidenbach MA, et al. (2012) Mapping yeast N-glycosites with isotopically recoded glycans. Mol Cell Proteomics 11(6):M111.015339

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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.

Reference Type
Journal Article
Authors
Breidenbach MA, Palaniappan KK, Pitcher AA, Bertozzi CR
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