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

开发一种大规模分析线虫中糖基化蛋白质的方法。

• 批准号: 16510148
• 负责人: KAJI Hiroyuki
• 金额: $ 1.98万
• 依托单位: Tokyo Metropolitan University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16510148/
• 关键词: C.elegans; lectin; glycoprotein; stable-isotope labeling; proteome; mass spectrometry; post-translational modification; shotgun; C.elegans; C.ELEGANS; LC-MS

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

本研究旨在利用线虫作为复杂蛋白混合物的来源，开发一种高通量和大规模分析糖基化蛋白的方法。蠕虫糖蛋白的大规模鉴定。在液体培养基中培养的蠕虫，用超声波破坏，并将其蛋白质分成可溶和不溶部分。每个蛋白组分用7M胍- hcl溶液溶解，用0.5 M Tris-HCl缓冲，用二硫苏糖醇还原，用碘乙酰胺烷基化。蛋白质经胰蛋白酶消化后，分别应用于三种凝集素亲和层析柱：conA、小麦胚芽凝集素（WGA）和蠕虫凝集素-6 （GaL6）。得到的糖肽在Sepharose柱上通过亲水性相互作用层析进一步纯化。用n -糖肽酶A在O -18稳定同位素标记水（H_2^<18>O）中处理糖肽。用2D-LC-MS/MS shotgun蛋白质组分析系统对标记的多肽进行分析。在MS/MS数据的基础上，以MASCOT为搜索引擎，利用虫pep序列数据集进行数据库检索。最后，共鉴定出830个n -糖蛋白，并确定了它们的糖基化位点。蠕虫糖蛋白的大规模定量分析。为了建立糖蛋白的定量分析方法，选择了一种引入质量标签的试剂，分别标记为C-12/C-13和N-14/N-15，并以鸡卵泡样蛋白为模型糖蛋白，测试了几种反应条件。卵泡样蛋白用胰蛋白酶消化，所得多肽混合物分为两部分。一种是用轻同位素的试剂，另一种是用重同位素的试剂。将两组分按一定比例混合，在0 -18白化水中用糖肽酶处理。采用nanoLC-MS/MS法对其进行分析，并对其糖肽进行了鉴定和相对定量。从质谱信号强度来看，在给定的混合比例下，糖肽可以相对定量。现在，使用蠕虫cona结合肽的复杂蛋白质混合物，整体程序的条件正在测试中。少