Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry

• DOI: 10.1021/pr300967y
• Publication Date: 2013-01-09T15:41:53Z
• Authors: Hannes Hahne; Nadine Sobotzki; Tamara Nyberg; Dominic Helm; Vladimir S. Borodkin; Daan M. F. van Aa...; Brian Agnew; Bernhard Kuster
• Keywords: Proteome; Proteolysis; Chemical Biology

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