title: Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.
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.
authors.affiliation: Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences, Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Hahne
authors.forename: Nadine
authors.initials: N
authors.lastname: Sobotzki
authors.forename: Tamara
authors.initials: T
authors.lastname: Nyberg
authors.forename: Dominic
authors.initials: D
authors.lastname: Helm
authors.forename: Vladimir S
authors.initials: VS
authors.lastname: Borodkin
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
authors.forename: Brian
authors.initials: B
authors.lastname: Agnew
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002853:Chromatography, Liquid; D057930:Click Chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000302:isolation & purification* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000480:Alkynes; D001386:Azides; D004791:Enzyme Inhibitors; D055767:Immobilized Proteins; D010446:Peptide Fragments; D020543:Proteome; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; D014357:Trypsin; D000117:Acetylglucosamine