期刊
JOURNAL OF PROTEOME RESEARCH
卷 12, 期 2, 页码 927-936出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr300967y
关键词
proteomics; mass spectrometry; post-translational modifications; O-GlcNAc; metabolic labeling; Click chemistry; beta-elimination
资金
- Studienstiftung des deutschen Volkes e. V.
- Faculty Graduate Center Weihenstephan of TUM Graduate School at the Technische Universitat Munchen, Germany
- MRC [G0900138] Funding Source: UKRI
- Medical Research Council [G0900138] Funding Source: researchfish
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 beta-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.
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