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

作为一个后基因组计划，蛋白质组分析是最重要的问题之一。通常，蛋白质组学分析通过参考基因组数据库确定目标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起作用的成熟蛋白。在本项目中，获得了以下结果：1。n端肽2分离与表征新方法的建立。含钌化合物n端标记试剂的研制及蛋白质络合物的分析方法蛋白质和多肽的选择性c端修饰及蛋白质组学分析新方法采用高效液相色谱法和自动定位仪对2-硝基苯磺酸基标记肽进行快速高效的MALDI-TOF质谱峰检测。首先，采用亲和生物素-生物素技术分离后，采用含有生物素和二硫基团的试剂进行选择性n端标记。二硫键被氧化裂解，释放出n端肽，用质谱分析得到氨基酸序列。二是钌络合物对蛋白质的选择性标记。酶解后，标记的n端片段不需要任何分离就能被完全检测到。将该试剂用于蛋白质混合物，可以同时检测组成蛋白质的所有n端。下一个是通过恶唑酮选择性标记c端。中间体，能够成功测定c端氨基酸序列。这些原创作品在项目中获得了多篇报告、国内外专利和商业化产品。

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.