Endo‐M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O‐GlcNAcylation Analysis

摘要

Abstract To selectively enrich O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O‐GlcNAc moieties are ligated with long N‐glycans using an Endo‐M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild‐type Endo‐M/S to restore the O‐GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag‐free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome‐wide analysis of protein O‐GlcNAcylation to promote its mechanism research.