Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging
Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging
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DOI：
10.1038/nprot.2006.444
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Isobe, Toshiaki
中科院分区：
文献类型：
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作者：
Kaji, Hiroyuki;Yamauchi, Yoshio;Isobe, Toshiaki
蛋白质的翻译后修饰（PTM），如糖基化和磷酸化，是各种信号和调控事件的关键，因此是蛋白质组学研究的一个重要目标。我们在这里描述了一个协议的同位素编码的糖基化位点特异性标记（IGOT），一种方法，用于大规模识别N-连接的糖蛋白从复杂的生物样品。该方法的步骤是：（1）凝集素柱介导的亲和捕获由蛋白质混合物的蛋白酶消化产生的糖肽;（2）通过亲水相互作用色谱（HIC）纯化富集的糖肽;（3）肽-N-聚糖酶介导的稳定同位素标签（18）O的掺入，特别是在N-糖基化位点;和（4）通过基于液相色谱-质谱联用（LC/MS）的蛋白质组学技术鉴定（18）0-标记肽。该协议的应用程序的N-连接的糖蛋白从线虫线虫或小鼠肝脏的粗提取物的特性提供了一个列表的数百至一千糖蛋白和它们的糖基化位点在一周内。
Protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, are crucial for various signaling and regulatory events, and are therefore an important objective of proteomics research. We describe here a protocol for isotopecoded glycosylation site-specific tagging (IGOT), a method for the large-scale identification of N-linked glycoproteins from complex biological samples. The steps of this approach are: (1) lectin column-mediated affinity capture of glycopeptides generated by protease digestion of protein mixtures; (2) purification of the enriched glycopeptides by hydrophilic interaction chromatography (HIC); (3) peptide-N-glycanase-mediated incorporation of a stable isotope tag, (18)0, specifically at the N-glycosylation site; and (4) identification of (18)0-tagged peptides by liquid chromatography-coupled mass spectrometry (LC/MS)-based proteomics technology. The application of this protocol to the characterization of N-linked glycoproteins from crude extracts of the nematode Caenorhabditis elegans or mouse liver provides a list of hundreds to a thousand glycoproteins and their sites of glycosylation within a week.