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Development of Highly Sensitive, Accurate, and Quantitative Analytical System for Proteome

开发高灵敏、准确、定量的蛋白质组分析系统

基本信息

批准号：
15201043
负责人：
OKAMURA Taka-aki
金额：
$ 32.53万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15201043/
关键词：
Proteome Metal Complex Protein Mass Analysis Amino Acid Sequencing N-terminal Peptide C-terminal Peptide Labeling Reagent N端ペプチド C端ペプチドビオチンプロテオミクスプロテオーム解析質量分析計 de novo sequencing オキサゾロン

项目摘要

As a post-genome project, proteome analysis is one of the most important problems. Generally, proteome analyses identify precursor proteins on genes by determining internal sequences of the target protein referred to genome data base. However, these methods can not determine accurately mature proteins working in important biological systems. In this project, the following results were obtained.1. Establishment of a new method for isolation and characterization of N-terminal peptide2. Development of N-terminal labeling reagent having ruthenium compound and analytical method of protein complexes using this reagent3. Selective C-terminal modification of proteins and peptides and new analytical methods for proteome4. Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatusThe first is selective N-terminal labeling by a reagent having biotin and disulfide moieties following isolation using avidin-biotin technique. The disulfide bond was oxidatively cleaved to release N-terminal peptide, which was analyzed by mass spectroscopy to give amino acid sequences. The second is selective labeling of proteins by ruthenium complex. After enzymatic digestion, the labeled N-terminal fragment was exclusively detected without requiring any separation. Using this reagent to a mixture of proteins afforded simultaneous detection of all the N-termini of constituent proteins. The next one is selective C-terminal labeling via oxazolone. intermediate, which enabled successful determination of C-terminal amino acid sequences. These original works in this project resulted in many reports, domestic and overseas patents, and commercialized products.

作为后基因组计划，蛋白质组分析是最重要的问题之一。一般来说，蛋白质组分析通过参考基因组数据库确定靶蛋白的内部序列来鉴定基因上的前体蛋白。然而，这些方法不能准确地确定在重要生物系统中工作的成熟蛋白质。在本项目中，取得了以下成果.一种新的N-端肽2分离鉴定方法的建立。开发具有钌化合物的N-末端标记试剂和使用该试剂的蛋白质复合物的分析方法3.蛋白质和肽的选择性C-末端修饰和蛋白质组的新分析方法4。高效液相色谱与自动点样仪联用快速、高效地检测2-硝基苯亚磺酰基标记肽的MALDI-TOF MS峰第一种方法是在用亲和素-生物素技术分离后，用含有生物素和二硫键的试剂选择性地标记N-末端。二硫键被氧化裂解以释放N-末端肽，通过质谱分析以得到氨基酸序列。二是钌配合物对蛋白质的选择性标记。酶消化后，标记的N-末端片段被专门检测而不需要任何分离。用该试剂对蛋白质混合物进行检测，可同时检测组成蛋白质的所有N-末端。下一个是通过恶唑酮的选择性C-末端标记。中间体，这使得能够成功地确定C-末端氨基酸序列。本项目的这些原创性工作产生了多项报告、国内外专利和商业化产品。

项目成果

期刊论文数量（552）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry

DOI：
10.1021/ac048776w
发表时间：
2005-01-15
期刊：
ANALYTICAL CHEMISTRY
影响因子：
7.4
作者：
Yamaguchi, M;Nakazawa, T;Norioka, S
通讯作者：
Norioka, S

Syntheses, structures, and luminescent and magnetic properties of novel three-dimensional lanthanide complexes with 1,3,5-benzenetriacetate.

DOI：
10.1021/ic050453a
发表时间：
2005-08
期刊：
Inorganic chemistry
影响因子：
4.6
作者：
Zheng-Hua Zhang;T. Okamura;Y. Hasegawa;H. Kawaguchi;Ling-yan Kong;Wei‐Yin Sun;N. Ueyama
通讯作者：
Zheng-Hua Zhang;T. Okamura;Y. Hasegawa;H. Kawaguchi;Ling-yan Kong;Wei‐Yin Sun;N. Ueyama

Trends in Proteome Analysis

蛋白质组分析的趋势