大鼠心肌线粒体蛋白质组学样本制备方法学的探讨

Study on the preparation method of myocardial mitochondrial proteomics samples in rats

目的寻找心肌线粒体蛋白质组学研究中样本制备关键环节的可行性方案。方法分为组织匀浆组(TH组)、差数离心提取心肌线粒体组(CM组)及差数离心后再通过Nycodenz密度梯度离心法提纯线粒体组(PM组)。通过Western blot法检测各亚细胞器标志性蛋白的含量验证线粒体的纯度,提取CM组与PM组中的蛋白质行双向凝胶电泳(2-DE),观察硝酸银染色后CM组与PM组的凝胶图像,选取PM组中的部分蛋白质斑点酶解后质谱鉴定。结果本实验提纯的线粒体纯度较高;PM组蛋白质2-DE后凝胶银染图像较CM组更清晰,背景更佳;选取的部分蛋白质经鉴定均为线粒体蛋白。结论本实验为心肌线粒体蛋白质组学研究中的样本制备提供了较好的方法。

Objective To explore the feasibility of the key link of sample preparation in myocardial mitochondrial proteomics.Methods It was divided into tissue homogenate group（group TH）and the differential centrifugation myocardial mitochondria group（group CM）and nycodenz density gradient centrifugation to purify the mitochondria group（group PM）.The content of various subcellular markers was detected by western blot（WB）to detect the purity of mitochondria.The mitochondrial proteins were extracted to two-dimensional electrophoresis（2-DE）.It was observed the gel images of group CM and group PM after silver staining.Some spots of group PM were identified after enzyme hydrolysis by mass spectrometry.Results The mitochondrial purity in group PM was higher than in group CM.The gel image and background of group PM after silver staining was clearer than group CM.The selected proteins were identified as mitochondrial proteins.Conclusion The study provides a better method for the preparation of the samples in myocardial mitochondrial proteomics.