人晶状体上皮细胞蛋白质组的双向电泳和质谱鉴定

The proteome research of human lens epithelial cells by two-dimensional gel electrophoresis and mass spectrometry

目的:建立人晶状体上皮细胞蛋白质组研究体系,探讨双向电泳和质谱鉴定技术在人晶状体上皮细胞蛋白质组研究中的作用。方法:体外培养人晶状体上皮细胞株,用两种不同方法提取总蛋白,进行固相pH梯度(IPG)等电聚焦双向凝胶电泳,凝胶通过GS-800扫描仪(Bio-Rad)获取图像并使用PDQuest专业图像分析软件分析。在此基础之上,胰酶消化蛋白质斑点并进行质谱分析。结果:获得了重复性较好的人晶状体上皮细胞蛋白质组电泳图谱。晶状体蛋白质斑点在等电点pH值为4-7、相对分子质量为17-72 kD之间均有分布。其中高丰度蛋白点主要分布于分子量19-50 kD、PI 5-7范围内。2个蛋白点通过质谱分析和数据库的检索得到了初步的鉴定。结论:建立起了一个稳定的分析人晶状体上皮细胞蛋白质组学实验体系;为进一步研究人类晶状体在生理状态及白内障等病理条件下的改变提供了蛋白质组学的研究方法和途径。

AIM： To establish the techniques in the proteome research of human lens epithelial cells, including the techniques of two - dimensional electrophoresis and mass spectrometry. METHODS ： Total protein of cultured human lens epithelial cells was extracted with two kinds of different methods. The proteins were separated using immobilized pH gradients 2 - DE and visualized by silver staining. The digitized images obtained by GS - 800 scaner were then analyzed with PDQuest software in order to establish the differential expression,profiles. The differential expressed protein spots were cut from the gels using proteomework spot cutter and subjected to in - gel digestion with trypsin. The digested peptide separation was conducted by a Finnigan LCQ MS coupled with a Surveyor HPLC system. RESULTS： A high resolution and reproducible 2 - ED image was successfully obtained. The maps of 2 - DE showed that lens proteins were in the section of pH 4 -7 which the relative molecular weight was 17 -72 kD. Relative molecular weight of more abundant proteins was localized at 19 -50 kD, as well as the isoelectric points were found to lie between PI 5 -7. Two of these proteins were identified by mass spectrometry and database queries. CONCLUSION： A stable protein maps of human lens epithelial cells is constructed. The technique will be used in human lens research to characterize physiological processes and diseases.