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CONSTRUCTION OF METHOD FOR PROTEOMICS RESEARCH

蛋白质组学研究方法的构建

基本信息

批准号：
12470485
负责人：
GOTO Junichi
金额：
$ 9.15万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Gene can display its function owing to translate correspond to protein, and the post-translational modification of protein is involved in a functional expression. Proteome research is a new field, which is starting to have a major impact in the post-genome era. For reconstruction of life system, proteomics focuses the structural and functional analysis of proteins, which exchange their expression dynamically and interact to other biological active substances.HeLa S3 cells were cultured with a medium containing 50 μM deoxycholic acid (DCA), and the effect of DCA stimulation to cells was investigated by the differential display. The clear protein expression change was observed in the cytosolic fraction, and the peptide fragments mixture was measured by MALDI-TOFMS on the heels of the in-gel digestion. According to the results using by peptide mass fingerprinting method, the interested protein was identified as peroxiredoxin I, which played an important role for the oxidative stress. On the other hand, an anti-DCA monoclonal antibody was produced using a DCA-bovine serum albumin immune complex. And it was immobilized to the agarose gel for production of immunoaffinity gel. The novel affinity labeling reagents, acyl adenylate and acyl 2-deoxyglucuronide, was developed for analysis of intramolecular interaction, and investigated their reactivity. The results indicated that the affinity labeling reagents developed could react selectively to interaction site on protein molecule by control of pH of reaction mixture. The peptide labeled DCA could be selectively extracted from enzyme digested peptide mixture using the immunoaffinity gel developed in this study.

基因通过翻译对应于蛋白质来发挥其功能，而蛋白质的翻译后修饰参与了功能表达。蛋白质组研究是后基因组时代开始产生重大影响的一个新领域。为了重建生命系统，蛋白质组学主要研究蛋白质的结构和功能，这些蛋白质的表达是动态交换的，并与其他生物活性物质相互作用。在胞质组分中观察到明显的蛋白表达变化，并且在胶内消化之后通过MALDI-TOFMS测量肽片段混合物。根据肽质量指纹图谱分析结果，将目的蛋白鉴定为过氧化物氧还蛋白I，该蛋白在氧化应激中起重要作用。另一方面，使用DCA-牛血清白蛋白免疫复合物制备抗DCA单克隆抗体。将其固定在琼脂糖凝胶上制备免疫亲和凝胶。开发了用于分子内相互作用分析的新型亲和标记试剂酰基腺苷酸和酰基2-脱氧葡萄糖醛酸苷，并研究了它们的反应性。结果表明，所研制的亲和标记试剂可通过控制反应液的pH值，选择性地与蛋白质分子上的相互作用位点发生反应。用免疫亲和凝胶从酶消化的肽混合物中选择性地提取肽标记的DCA。