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.

作为基因组后项目，蛋白质组分析是最重要的问题之一。通常，蛋白质组分析通过确定所指基因组数据库的靶蛋白的内部序列来鉴定基因上的前体蛋白。但是，这些方法无法确定在重要生物系统中起作用的成熟蛋白质。在此项目中，获得以下结果1。建立一种新方法，用于隔离和表征N末端肽2。使用该试剂的N端标记试剂的开发具有氟化合物和蛋白质复合物的分析方法的开发3。蛋白质和肽的选择性C末端修饰以及蛋白质组的新分析方法。快速有效的MALDI-TOF MS峰值检测2-硝基苯苯基标记的肽使用HPLC和自动斑点仪的组合使用，首先是通过使用Avidin-Biotin-Biotin技术隔离的生物素和二硫化物部分的试剂选择性N端标记。将二硫键在氧化中裂解以释放N末端肽，通过质谱法分析了氨基酸序列。第二个是通过ruthenium复合物对蛋白质进行选择性标记。酶消化后，仅检测到标记的N末端片段而无需任何分离。使用该试剂与蛋白质混合物，可同时检测所有组成蛋白的N末端。下一个是通过恶唑酮选择性的C末端标记。中级，能够成功地测定C末端氨基酸序列。该项目中的这些原创作品产生了许多报告，国内和海外专利和商业化产品。