Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry
Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry
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结合多种酶消化和酰肼化学对人体肝脏组织进行糖蛋白组学分析
DOI：
10.1021/pr8008012
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发表时间：
2009-02-01
影响因子：
4.4
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
--
作者：
Chen, Rui;Jiang, Xinning;Zou, Hanfa
由于糖蛋白极其复杂、动态范围分布广、化学计量修饰少，糖基化的研究远远落后于当前蛋白质组学的进展。利用肼化学方法固相萃取胰蛋白酶n -糖肽已成为分析n -糖蛋白组的常用方法。然而，胰蛋白酶对人类蛋白质组数据库中蛋白质的硅酶切表明，有很大比例的胰蛋白酶n -糖肽不在鸟枪法蛋白质组学方法的优选检测质量范围内，即800 - 3500 Da。较大的糖基可能会阻碍胰蛋白酶进入n -糖苷基附近的K、R残基进行消化，从而产生较大的糖肽。因此，如果仅使用胰蛋白酶来消化蛋白质，许多n -糖苷位点无法定位。本文介绍了一种结合肼化学法和多种酶切法分析人肝组织n -糖蛋白组的综合方法。将人肝组织裂解物分别用三种不同特异性的蛋白酶（胰蛋白酶、胃蛋白酶和热溶酶）进行酶解。单独使用胰蛋白酶鉴定了622个n -糖苷位，而使用胃蛋白酶和热溶酶鉴定了另外317个n -糖苷位。在317个n -糖苷位点中，98个（30.9%）在理论上不能被胰蛋白酶识别，因为相应的硅胰蛋白酶肽要么太小，要么太大，无法用质谱仪检测。本研究清楚地表明，由于采用多种酶消化，n -糖苷的覆盖范围可以显著增加。共鉴定出939个n -糖位点，覆盖了523个来自人肝组织的无冗余糖蛋白，从而建立了迄今为止最大的人肝糖蛋白组数据集。
The study of protein glycosylation has lagged far behind the progress of current proteomics because of the enormous complexity, wide dynamic range distribution and low stoichiometric modification of glycoprotein. Solid phase extraction of tryptic N-glycopeptides by hydrazide chemistry is becoming a popular protocol for the analysis of N-glycoproteome. However, in silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. Herein, we describe a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion. The lysate of human liver tissue was digested with three proteases, that is, trypsin, pepsin and thermolysin, with different specificities, separately. Use of trypsin alone resulted in identification of 622 N-glycosites, while using pepsin and thermolysin resulted in identification of 317 additional N-glycosites. Among the 317 additional N-glycosites, 98 (30.9%) could not be identified by trypsin in theory because the corresponding in silico tryptic peptides are either too small or too big to detect in mass spectrometer. This study clearly demonstrated that the coverage of N-glycosites could be significantly increased due to the adoption of multiple enzyme digestion. A total number of 939 N-glycosites were identified confidently, covering 523 noredundant glycoproteins from human liver tissue, which leads to the establishment of the largest data set of glycoproteome from human liver up to now.