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。

基本信息

  • 批准号:
    16510148
  • 负责人:
  • 金额:
    $ 1.98万
  • 依托单位:
  • 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

项目摘要

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
本研究旨在开发一种使用线虫作为复杂蛋白质混合物来源对糖基化蛋白质进行高通量和大规模分析的方法。1.大规模鉴定线虫糖蛋白。线虫,在液体培养基中培养,通过超声破碎,将其蛋白质分离成可溶性和不溶性级分,每种蛋白质级分用7M盐酸胍缓冲溶液溶解。用0.5 M Tris-HCl,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、麦芽凝集素(WGA)和蠕虫。 glectin-6 (GaL6) 通过亲水相互作用色谱进一步纯化。糖肽柱在 o-18 稳定同位素标记水 (H_2^<18>O) 中用 N-糖肽酶 A 处理,并通过 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序列数据集进行数据库搜索,最终鉴定出总共830个蛋白质。确定了N-糖蛋白及其糖基化位点。2.对线虫糖蛋白进行大规模定量。为了开发一种糖蛋白定量分析方法,引入了C-差异标记的质量标签。选择 12/C-13 和 N-14/N-15,并使用鸡卵类粘蛋白作为模型糖蛋白进行消化,测试了几种反应条件。将胰蛋白酶和所得肽混合物分离成两个级分,一个用具有轻同位素的试剂标记,另一个用具有重同位素的试剂标记,这两个级分以一定的比例混合,并用糖肽酶在o-中处理。采用nanoLC-MS/MS方法对18个标记的水进行分析,并根据MS信号强度对糖肽进行鉴定和相对定量。现在,使用蠕虫 conA 结合肽的复杂蛋白质混合物,对整个程序的条件进行了测试。

项目成果

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KAJI Hiroyuki其他文献

KAJI Hiroyuki的其他文献

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{{ truncateString('KAJI Hiroyuki', 18)}}的其他基金

Automatic Construction of a Sense-disambiguated Multilingual Dictionary
语义消歧多语言词典的自动构建
  • 批准号:
    22300032
  • 财政年份:
    2010
  • 资助金额:
    $ 1.98万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)

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