一种规模化蛋白质组分离和鉴定新方法研究及其应用

Studies on a Novel and Large-scale Proteomics Method and Its Application

建立了一种规模化的蛋白质组分离和鉴定新方法。通过对在生命发育过程中具有重要研究价值的人胎肝线粒体蛋白质组的分离分析,表明与毛细管液-质联用的不同分离方法的组合可以增大检测动态范围和分辨率。研究共鉴定了2977个肽段,归属于915种蛋白质。去除批次间冗余后,鉴定的蛋白质为477种,其中291种为唯一蛋白质,186种为蛋白质簇,144种蛋白质明确定位于人胎肝线粒体中。所鉴定蛋白质的分子量分布范围为7000 Da^330000 Da,pI值分布在4.0~11.89,克服了两维凝胶电泳在分子量和pH方面的歧视性问题。实验中发现的蛋白质簇以及确定一种蛋白质需要最少肽段数的问题还需要进一步研究。

A novel and large-scale proteomic method has been established and applied for profiling mitochondria proteome of human fetal liver. The experimental results showed that combination of different orthogonal separation methods followed by capillary reversed-phase liquid chromatography and on-line eiectrospray ionization tandem mass spectrometry expanded the dynamic range and resolution, and enabled the identification of 915 proteins from 2977 different peptides. After deletion of redundant proteins among different batches of data, 477 proteins were identified, in which 291 proteins were unique proteins and 186 proteins were cluster proteins. 144 proteins were clearly localized in mitochondria of human fetal liver. The molecular weights of the identified proteins ranged from 7000 Da to 330000 Da and pI values from 4.0 to 11.89. These results demonstrated that liquid phase based separation could overcome some shortcomings adherent to two-dimensional gel-electrophoesis. However, some common issues, such as protein clusters or protein groups and peptide number limited for a reliable protein identification existed in both gel based separation and liquid phase based separation, still have to be addressed in the future researches.