Quantitative Profiling of Protein O-GlcNAcylation Sites by an Isotope-Tagged Cleavable Linker

ACS Chem Biol. 2018 Aug 17;13(8):1983-1989. doi: 10.1021/acschembio.8b00414. Epub 2018 Jul 30.


Large-scale quantification of protein O-linked β- N-acetylglucosamine (O-GlcNAc) modification in a site-specific manner remains a key challenge in studying O-GlcNAc biology. Herein, we developed an isotope-tagged cleavable linker (isoTCL) strategy, which enabled isotopic labeling of O-GlcNAc through bioorthogonal conjugation of affinity tags. We demonstrated the application of the isoTCL in mapping and quantification of O-GlcNAcylation sites in HeLa cells. Furthermore, we investigated the O-GlcNAcylation sensitivity to the sugar donor by quantifying the levels of modification under different concentrations of the O-GlcNAc labeling probe in a site-specific manner. In addition, we applied isoTCL to compare the O-GlcNAcylation stoichiometry levels of more than 100 modification sites between placenta samples from male and female mice and confirmed site-specifically that female placenta has a higher O-GlcNAcylation than its male counterpart. The isoTCL platform provides a powerful tool for quantitative profiling of O-GlcNAc modification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / chemistry*
  • Animals
  • Biotin / analogs & derivatives
  • Carbon Isotopes
  • Female
  • Glycoproteins / analysis*
  • Glycoproteins / chemistry
  • Glycosylation
  • HeLa Cells
  • Humans
  • Isotope Labeling / methods*
  • Male
  • Mice
  • Molecular Probes / chemistry*
  • Nitrogen Isotopes
  • Proteome / analysis*
  • Proteome / chemistry
  • Proteomics / methods*
  • Triazoles / chemistry


  • Carbon Isotopes
  • Glycoproteins
  • Molecular Probes
  • Nitrogen Isotopes
  • Nitrogen-15
  • Proteome
  • Triazoles
  • Biotin
  • Carbon-13
  • Acetylglucosamine