Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins
Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins
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DOI：
10.1038/nbt829
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发表时间：
2003-06-01
影响因子：
46.9
通讯作者：
Isobe, T
中科院分区：
文献类型：
--
作者：
Kaji, H;Saito, H;Isobe, T
我们在此描述了一种从复杂生物样品中大规模鉴定 N-糖基化蛋白质的策略。该方法被称为同位素编码糖基化位点特异性标记 (IGOT)，基于凝集素柱介导的对蛋白质混合物的胰蛋白酶消化产生的一组糖肽的亲和捕获，然后通过肽-N-糖苷酶介导将稳定同位素标签 O-18 特异性地掺入 N-糖基化位点。然后通过基于多维液相色谱-质谱 (LC-MS) 的技术鉴定 O-18 标记的肽。应用该方法对秀丽隐杆线虫蛋白提取物中的 N-连接高甘露糖和/或混合型糖蛋白进行表征，可以鉴定 250 种糖蛋白，包括 83 种假定的跨膜蛋白，同时测定 400 个独特的 N-糖基化位点。由于该方法适用于广泛的糖蛋白的系统鉴定，因此它应该促进基础糖生物学研究，并且可能可用于诊断应用，例如疾病相关糖蛋白的全基因组筛查。
We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, O-18, specifically into the N-glycosylation site. The O-18-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.