Enrichment of O-GlcNAc modified proteins by the periodate oxidation-hydrazide resin capture approach.
Enrichment of O-GlcNAc modified proteins by the periodate oxidation-hydrazide resin capture approach.
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DOI：
10.1021/pr900984h
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发表时间：
2010-05-07
影响因子：
4.4
通讯作者：
Medzihradszky, Katalin F.
中科院分区：
文献类型：
--
作者：
Klement, Eva;Lipinszki, Zoltan;Kupihar, Zoltan;Udvardy, Andor;Medzihradszky, Katalin F.
关键词：
已经开发了用于富集O-GlcNAc修饰的蛋白质的化学衍生化方法。该程序是基于分离技术用于N-糖蛋白与适当的修改，因为在两种类型的糖基化的差异：一个长期的高碘酸氧化，其次是酰肼树脂捕获，树脂上的蛋白水解消化和释放的修饰肽的羟胺。这种富集策略在质谱分析中提供了额外的好处。在碰撞活化时，开放的糖环的存在导致特征性片段化，从而促进糖肽鉴定和位点分配。将富集方案应用于先前描述为O-GlcNAc修饰的果蝇蛋白酶体复合物。O-GlcNAc修饰位于蛋白酶体相互作用蛋白、去泛素化酶Faf（CG 1945）和含有泛素样结构域的蛋白（CG 7546）上。还发现了其他三种GlcNAc修饰的蛋白质，HSP 70同源物（CG 2918），scribbled（CG 5462）和205 kDa微管相关蛋白（CG 1483）。有趣的是，在HSP 70同源物中，GlcNAc修饰连接到N-糖基化基序的天冬酰胺残基上。
A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.
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