Development of a method for large-scale analysis of glycosylated proteins in the worm, C.elegans.

本研究以秀丽隐杆线虫（C.elegans）为研究对象，建立了一种高通量、大规模分析线虫糖基化蛋白的方法。1.线虫糖蛋白的大规模鉴定将培养在液体培养基中的蠕虫进行超声破碎，将其糖蛋白分离为可溶性和不溶性部分。将每种蛋白质级分用0.5 M Tris-HCl缓冲的7 M胍-HCl溶液溶解，用二硫苏糖醇还原，并用碘乙酰胺烷基化。用胰蛋白酶消化蛋白质，并将等分试样分别应用于三种类型的凝集素亲和层析柱，conA，麦胚凝集素（WGA）和蠕虫凝集素-6（GaL 6）。通过Sepharose柱上的亲水相互作用层析进一步纯化所获得的糖肽。糖肽在~（18）O稳定同位素标记水（H_2^ O）中用N-糖肽酶A处理<18>。标记的肽 ...更多信息 采用2D-LC-MS/MS鸟枪法蛋白质组分析系统进行分析。基于MS/MS数据，使用MASCOT作为搜索引擎和wormpep序列数据集通过数据库搜索来鉴定肽。最终鉴定出830个蛋白质为N-糖蛋白，并对其糖基化位点进行了测定.蠕虫糖蛋白的大规模定量为了建立蠕虫糖蛋白的定量分析方法，选择了一种引入质量标签的试剂，并以鸡卵类粘蛋白为模型糖蛋白，对几种反应条件进行了试验。用胰蛋白酶消化卵类粘蛋白，并将所得肽混合物分离成两个级分。一种是用轻同位素试剂，另一种是用重同位素试剂。将这两种级分以一定比例混合，并在0 -18倍的水中用糖肽酶处理。通过nanoLC-MS/MS方法分析肽，并对其糖肽进行鉴定和相对定量。根据MS信号强度，可以在给定的混合比例下相对定量糖肽。现在，使用蠕虫conA结合肽的复杂蛋白质混合物，测试整个程序的条件。少

This research was aimed at the development of a method for high-throughput and large-scale analysis of glycosylated proteins using the worm C.elegans as a source of complex protein mixture.1.Large-scale indentification of the worm glycoproteins.The worms, which were cultured in liquid medium, were disrupted by sonication and their proteins were separated into the soluble and insoluble fractions. Each protein fractions were dissolved with 7M guanidine-HCl solution buffered with 0.5 M Tris-HCl, reduced with dithiothreitol, and alkylated with iodo acetoamide. The proteins were digested with trypsin and the aliquots were separately applied to three types of lectin affinity chromatography columns, conA, wheat germ agglutinin (WGA), and the worm glectin-6 (GaL6). Obtained glycopeptides were further purified by hydrophilic interaction chromatography on Sepharose column. The glycopeptides were treated with N-glycopeptidase A in o-18 stable-isotope lebeled water (H_2^<18>O). The labeled peptide … More s were analyzed by 2D-LC-MS/MS shotgun proteome analysis system. Based on the MS/MS data, the peptides were identified by database searching using MASCOT as search engine and wormpep sequence dataset. Finally, total 830 proteins were identified as N-glycoproteins and their glycosylated sites were determined.2. Large-scale quantification of the worm glycoproteins.In order to develop a method for quantitative analysis of the glycoproteins, a reagent to introduce a mass-tag, which is differentially labeled with C-12/C-13 and N-14/N-15, was sellected and several reaction conditions were tested using chicken ovomucoid as a model glycoprotein. Ovomucoid was digested with trypsin and the resultant peptide mixture was separated into two fractions. One was lebeled with the reagent with light isotopes, and the other was with the reagent with heavy isotopes. The two fractions were mixed at some given ratio, and treated with glycopeptidase in o-18 lebeled water. The peptides were analyzed by nanoLC-MS/MS method and its glycopeptides were identified and relatively quantified. From the MS signal intensity, the glycopeptides could be relatively quantified at given mixd ratio. Now, using a complex protein mixture of the worm conA-bound peptides, conditions for overall procedures are testing. Less