蛋白质组学技术分离及鉴定人脑氧化蛋白质

背景：氧化修饰蛋白质在脑老化及阿尔茨海默病等老年变性神经病的发病机制中扮演着重要角色。蛋白质组学技术可以发现氧化修饰蛋白质，为相关疾病的药物治疗选择新的靶标。 目的：建立以双向电泳结合Western blot及生物质谱技术分离、鉴定人脑氧化修饰蛋白质的方法。 方法：取3个无神经系统疾患的男性脑组织蛋白，第一向等电聚焦电泳后，固相pH梯度胶条同二硝基苯肼反应，然后行第二向SDS-PAGE电泳。2张相同凝胶中的1张行考马斯亮蓝染色，另1张凝胶上免疫荧光显色。同PVDF膜上显色点相对应的考染凝胶上的蛋白质点即为氧化修饰蛋白质。凝胶蛋白胶内酶解，基质辅助激光解析飞行时间串联质谱鉴定氧化修饰蛋白质。 结果与结论：从提取的脑组织中鉴定出β-肌动蛋白、天冬氨酸氨基转移酶、果糖二磷酸醛缩酶、磷酸甘油酸激酶、1，3-磷酸甘油醛脱氢酶、碳酰还原酶1、磷酸丙糖异构酶、转酮醇酶、丙酮酸激酶、血清白蛋白前体、二氢嘧啶酶相关蛋白质2、热休克蛋白60为氧化修饰蛋白质。说明实验成功建立了用蛋白质组学技术分离及鉴定人脑氧化蛋白质的方法。

Oxidatived proteins in human brains separated and identified by proteomics technology

BACKGROUND:Oxidize-modified proteins play an important role in the pathogenesis of Alzheimer’s disease (AD) and other geriatric diseases. These marker proteins can be detected using proteomics technology, which provide new drug targets for the treatment of related diseases. OBJECTIVE:To establish a method for separating and identifying human oxidatived proteins by using two-dimensional electrophoresis (2-DE), Western blot and mass spectrometry (MS). METHODS:The brains of subjects who died without clinical or pathological involvement of nervous system were obtained at autopsy. Following sample proteins isoelectric focusing (IEF), the immobilized pH gradient (IPG) strips were incubated in       10 mmol/L DNPH. Then sample IPG strips were prepared for SDS-PAGE electrophoresis. Proteins in one gel were Coomassie stained, and proteins from the other duplicate gel were eletroblotted to PVDF membrane using the ISO-DALT system. The PVDF membrane were incubated with the solution consisting of the anti-DNP antibody, and then incubated with the HRP. A DAB kit was used to visualize the immunostained blots. Protein spots of interest were excised from the gel, digested in situ with trypsin and identified using MALDI-TOF mass spectrometry or MALDI-TOF/TOF tandem mass spectrometry. RESULTS AND CONCLUSION:Oxidatived proteins were identified to be β-actin, aspartate aminotransferase, fructose-bisphosphate aldolase A, phosphoglycerate kinase 1, glyceraldehyde 3-phosphate dehydrogenase, carbonyl reductase 1, triosephosphate isomerase, carbonate dehydratase 1, similar to transketolase, pyruvate kinase, serum albumin precursor, dihydropyrimidinase-related protein 2 and heat shock 60 kD protein. The oxidatived proteins are quite useful for discovering the molecular mechanisms of AD and other geriatric diseases.