O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry
O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry
复制标题
结合化学酶标记、无铜点击化学、还原裂解和电子转移解离质谱进行 O-GlcNAc 位点定位
DOI：
10.1021/acs.analchem.8b05688
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发表时间：
2019-02-19
影响因子：
7.4
通讯作者：
Hart, Gerald W.
中科院分区：
文献类型：
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作者：
Ma, Junfeng;Wang, Wei-Han;Hart, Gerald W.
作为动态翻译后修饰，O-连接的β-N-乙酰葡糖胺（O-GlcNAc）修饰（即，O-GlcNAc酰化）调节许多涉及细胞代谢和信号传导的生物学过程。然而，O-GlcNAc位点定位，位点特异性功能表征的先决条件，一直是一个挑战，因为它的发现。在此，我们提出了一种新的方法，O-GlcNAc富集和位点定位。在该方法中，用UDP-GalNAz标记肽上的O-GlcNAc部分，然后用带有末端环辛炔、二硫桥和生物素柄的多功能试剂进行无铜叠氮化物-炔环加成。标记的肽然后在还原剂处理后从NeutrAvidin珠释放，用（3-丙烯酰胺丙基）三甲基氯化铵烷基化，并进行电子转移解离质谱分析。在通过使用标准合成肽gCTD和模型蛋白α-晶状体蛋白进行验证后，将这种方法应用于过表达的TGF-β激活的激酶1/MAP 3 K7结合蛋白2（TAB 2）的位点定位，其中明确鉴定了四个O-GlcNAc位点。我们的方法提供了一个很有前途的工具，为位点特异性表征的O-GlcNAc重要蛋白质。
As a dynamic post-translational modification, O-linked beta-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein alpha-crystallin, such an approach was applied to the site mapping of overexpressed TGF-beta-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.