CONSTRUCTION OF METHOD FOR PROTEOMICS RESEARCH

基因通过翻译对应蛋白质来发挥其功能，而蛋白质的翻译后修饰又涉及到一种功能表达。蛋白质组研究是一个新的领域，在后基因组时代开始产生重大影响。为了重建生命系统，蛋白质组学侧重于蛋白质的结构和功能分析，这些蛋白质可以动态地交换它们的表达并与其他生物活性物质相互作用。将HeLa S3细胞培养在含有50μM脱氧胆酸的培养液中，通过差异显示来观察脱氧胆酸对细胞的刺激作用。在胞浆部分观察到明显的蛋白表达变化，凝胶内消化后用MALDI-TOFMS检测多肽碎片混合物。根据多肽质量指纹法的结果，鉴定了目的蛋白为过氧化还蛋白I，它在氧化应激中发挥了重要作用。另一方面，利用DCA-牛血清白蛋白免疫复合物制备了抗DCA的单抗。并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--酰基腺苷和2-脱氧葡萄糖醛酸脂，并考察了它们的反应性。结果表明，所研制的亲和标记剂可以通过控制反应混合物的pH选择性地与蛋白质分子上的相互作用部位发生反应。利用本研究开发的免疫亲和凝胶，可以从酶消化的多肽混合物中选择性地提取标记DCA的多肽。

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.