Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.
Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.
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DOI：
10.1021/pr300967y
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发表时间：
2013-02-01
影响因子：
4.4
通讯作者：
Kuster B
中科院分区：
文献类型：
--
作者：
Hahne H;Sobotzki N;Nyberg T;Helm D;Borodkin VS;van Aalten DM;Agnew B;Kuster B
N-乙酰葡糖胺（O-GlcNAc）对蛋白质的翻译后修饰参与多种细胞过程的调节，并与许多慢性疾病相关。尽管其新兴的生物学意义，O-GlcNAc蛋白的系统鉴定仍然具有挑战性。在本研究中，我们证明了一个显着改进的O-GlcNAc蛋白富集程序，利用叠氮化物修饰的GlcNAc和铜介导的点击化学修饰的蛋白质的纯化的炔树脂上的细胞的代谢标记。使用胰蛋白酶进行树脂上蛋白水解，然后进行LC-MS/MS，从单个细胞系中鉴定出约1500个O-GlcNAc蛋白。随后使用选择性β-消除洗脱共价树脂结合的O-GlcNAc肽，使得能够鉴定82种蛋白质上的192个O-GlcNAc修饰位点。为了证明所开发的方法的实际效用，我们研究了O-GlcNAc酶抑制剂GlcNAc抑制素G对细胞蛋白的O-GlcNAc修饰水平的全局影响。约200种蛋白质，包括参与己糖胺信号传导途径的几种关键参与者，对药物的反应显示出显著增加的O-GlcNAc化水平，这进一步加强了O-GlcNAc蛋白质修饰与细胞营养感测和反应的联系。
The posttranslational 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 labelling 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 around1500 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 192 O-GlcNAc modification sites on 82 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 signalling 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.