Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry
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- 作者：Hannes Hahne ; Nadine Sobotzki ; Tamara Nyberg ; Dominic Helm ; Vladimir S. Borodkin ; Daan M. F. van Aalten ; Brian Agnew ; Bernhard Kuster
- 刊名：Journal of Proteome Research
- 出版年：2013
- 出版时间：February 1, 2013
- 年：2013
- 卷：12
- 期：2
- 页码：927-936
- 全文大小：413K
- 年卷期：v.12,no.2(February 1, 2013)
- ISSN：1535-3907
文摘
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鈥揗S/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 尾-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.