title: Mapping yeast N-glycosites with isotopically recoded glycans.
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.
authors.affiliation: Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Breidenbach
authors.forename: Krishnan K
authors.initials: KK
authors.lastname: Palaniappan
authors.forename: Austin A
authors.initials: AA
authors.lastname: Pitcher
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016384:Consensus Sequence; D006031:Glycosylation; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011134:Polysaccharides; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D000117:Acetylglucosamine