Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags

Nat Chem Biol. 2010 Sep;6(9):645-51. doi: 10.1038/nchembio.412. Epub 2010 Jul 25.


Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / analysis*
  • Acetylglucosamine / chemistry
  • Acetylglucosamine / metabolism*
  • Glycosylation
  • Kinetics
  • Mass Spectrometry
  • Methyl-CpG-Binding Protein 2 / genetics
  • Methyl-CpG-Binding Protein 2 / metabolism
  • Phosphorylation
  • Polyethylene Glycols / analysis
  • Polyethylene Glycols / chemistry*
  • Protein Processing, Post-Translational* / genetics
  • Transcription, Genetic


  • MECP2 protein, human
  • Methyl-CpG-Binding Protein 2
  • Polyethylene Glycols
  • Acetylglucosamine

Associated data

  • PubChem-Substance/96026614
  • PubChem-Substance/96026615
  • PubChem-Substance/96026616
  • PubChem-Substance/96026617
  • PubChem-Substance/96026618