Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry

J Proteome Res. 2013 Feb 1;12(2):927-36. doi: 10.1021/pr300967y. Epub 2013 Jan 18.


The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Alkynes / chemistry
  • Azides / chemistry
  • Chromatography, Liquid
  • Click Chemistry
  • Enzyme Inhibitors / pharmacology
  • HEK293 Cells
  • Humans
  • Immobilized Proteins / isolation & purification*
  • Immobilized Proteins / metabolism*
  • Peptide Fragments / analysis*
  • Protein Processing, Post-Translational*
  • Proteome / isolation & purification*
  • Proteome / metabolism*
  • Tandem Mass Spectrometry
  • Trypsin / chemistry
  • beta-N-Acetylhexosaminidases / antagonists & inhibitors
  • beta-N-Acetylhexosaminidases / metabolism


  • Alkynes
  • Azides
  • Enzyme Inhibitors
  • Immobilized Proteins
  • Peptide Fragments
  • Proteome
  • hexosaminidase C
  • beta-N-Acetylhexosaminidases
  • Trypsin
  • Acetylglucosamine