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

Angew Chem Int Ed Engl. 2022 Jun 7;61(23):e202117849. doi: 10.1002/anie.202117849. Epub 2022 Mar 30.


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.

Keywords: Chemoenzymatic Labeling; Glycopeptide Enrichment; Glycoproteomics; Hydrophilic Interaction Liquid Chromatography; O-GlcNAcylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism
  • Chromatography, Liquid / methods
  • Glycopeptides* / chemistry
  • Polysaccharides / chemistry
  • Proteome / analysis
  • Proteomics*


  • Glycopeptides
  • Polysaccharides
  • Proteome
  • Acetylglucosamine