液质联用多反应监测法定量肝纤维化非实质细胞中的ATPA蛋白质

MRM Quantifying ATPA in Nonparenchymal Cell from Liver Fibrosis

目的建立液质联用多反应监测方法定量复杂样品中的蛋白质。方法合成标准多肽,建立较通用的蛋白质多肽定量方法;挑选目标蛋白质及其定量离子对。采用SDS-PAGE方法分离复杂样品中的蛋白质,切取目标蛋白质条带进行胶内酶解和液质联用MRM定量,运用定量PCR方法分析目标蛋白在蛋白质和mRNA水平的一致性。结果选择在二维凝胶电泳中具有3倍差异的ATPA蛋白质进行了MRM定量分析,ATPA的4条多肽513.8、586.4、644.5和777.3对应的10个离子对均能特异地被质谱MRM方法检测,与正常对照相比,酒精性肝纤维化样本中ATPA的10个离子的曲线下面积变化基本一致,均表现为上调。MRM定量结果也表明ATPA在酒精性肝纤维化样品中上调了3.19倍,与二维凝胶电泳检测结果一致。进一步的定量PCR分析结果显示,该蛋白质的表达变化同步发生在mRNA和蛋白质水平。结论本研究构建了一种简单有效的液质联用MRM定量SDS-PAGE分离的复杂样品中蛋白质的方法。

Objective To establish mass spectrometer with multiple reaction monitoring quantification （ MRM ） method for target protein in complex samples. Methods By synthesis of polypeptide standards, we established a general quantitative method. According to the requirements,we selected the target protein and its quantitative ion pair. The complex protein samples was separated by SDS -PAGE. Then the target protein bands were cut and digested, the quantification of target protein was detected by mass spectrometry with MRM meth- od. we used quantitative PCR method to analyze the consistency of target proteins in the protein and mRNA levels. Results Four poly- peptides of ATPA were specifically detected by MRM method. Compared with the normal control, the areas under the curve which repre- sented 10 ion pairs of ATPA in alcoholic liver fibrosis were basically the same. MRM quantitative results that ATPA was up to 3.19 times were consistent with 2DE＇s in the sample of alcoholic liver fibrosis. The results of quantitative PCR analysis further showed that the chan- ges of ATPA occurred simultaneously at mRNA and protein levels. Conclusion We successfully established a simple and effective MRM quantitative method for complex protein samples.