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.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|