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

Citations Over TimeTop 10% of 2013 papers

Abstract

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.

Related Papers

• → Azide-Modified Graphitic Surfaces for Covalent Attachment of Alkyne-Terminated Molecules by “Click” Chemistry(2007)207 cited
• → Analysis and Control of Proteolysis of Recombinant Proteins in Escherichia coli(2004)60 cited
• → Phthalonitriles Functionalized for Click Chemistry. Design, Synthesis and Structural Characterization(2013)14 cited
• → Synthesis of Alkyne-Terminated PCDA Linker for Applying Click Chemistry on PDA Layers(2010)2 cited
• Proteolysis in alfalfa silage and its inhibiting method(2005)