应用超高分辨双向凝胶电泳技术进行正常人类肝脏蛋白质组研究  

Towards a High Resolution 2-DE Profile of Normal Human Liver Proteome Using Ultra-Zoom Gel

作　　者：米薇[1] 刘新[2] 贾伟[2] 李蕾[2] 蔡耘[2] 应万涛[2] 钱小红[2] 

机构地区：[1]中国计量科学研究院生物能源环境所,北京100013 [2]军事医学研究院放射与辐射医学研究所北京蛋白质组研究中心,蛋白质组学国家重点实验室,北京102206

出　　处：《中国科学：生命科学》2010年第9期843-852,共10页Scientia Sinica(Vitae)

基　　金：国家自然科学基金重点项目(批准号:30621063;20635010和20735005);国家重点基础研究发展计划(批准号:2006CB910801;2004CB518707和2007CB914100);国家高技术研究发展计划(批准号:2006AA02A308和2008AA02Z309)资助项目

摘　　要：肝脏是人体最大的实质器官,承担着人体许多关键的生理功能,在生命活动中占有重要地位.根据人类肝脏蛋白质组计划,本实验旨在构建人肝脏蛋白质双向凝胶电泳表达谱,尽可能分离和鉴定更多的蛋白.在双向凝胶电泳第一向等电聚焦水平上,从上样方式、水化液配方、聚焦时间等方面优化了碱性蛋白的分离条件,利用超放大胶分离技术搭建了高分辨的双向凝胶电泳分离平台,构建了人类肝脏蛋白质2-DE参考谱,检测到5481个蛋白质点.这是目前国际上最为全面的人体器官蛋白质组2-DE参考谱,为其他肝病的研究提供了较好的参照系.成功鉴定了429个非冗余蛋白,对pH4.0～7.0,5.0～6.0,5.5～6.7,6.0～9.0部分蛋白质点在胶上进行了注释,由此构建了人肝2-DE蛋白质表达谱数据库.对蛋白质的理化性质、功能及亚细胞定位进行了全面分析.研究中构建的人类肝脏蛋白质2-DE图谱将为肝脏比较蛋白质组学研究提供参考.Human liver is the largest organ in the body. It takes on many important physiological functions. The global analysis of human liver proteins is essential for a better understanding of the molecular basis of liver and its diseases. Under the coordination of Human Liver Proteome Project （HLPP）, the goal of the present study was to visualize and detect as many proteins as possible in normal human liver using two-dimensional gel electrophoresis and make a reference map of human normal liver proteins for the comprehensive analysis of human liver proteome and the other related research. For this purpose, Narrow pH range ultra-zoom 2-DE gels were developed and optimized to improve the resolution and enhance the detection of low abundance proteins. High resolution patterns of human liver in pH gradients 4.5-5.5, 5.0-6.0, 5.5-6.7, 6.0-9.0 and 6.0-11.0 were presented. To tackle the poor resolution in the alkaline pH range of 2-DE gels such as pH6-9 and pH6-11, we have optimized isoelectric focusing protocol including sample application using cup-loading at the anode and incorporation of 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. With the application of optimized protocol we get reproducible better resolution both in analytical and preparative 2-DE gels. The ultra-zoom gels pH4.5-9 revealed a total of 5481 protein spots compared to 2386 protein spots in 3-10 pH gradient and 1878 protein spots in 4-7 pH gradient. It is hoped that the visualized reference map of human liver proteins presented in this paper will be valuable for comparative proteomics of liver proteome research.

关 键 词：人类肝脏蛋白质组 双向凝胶电泳 超高分辨胶 质谱 

分 类 号：Q593.1[生物学—生物化学]