Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels  

作　　者：MI Wei LIU Xin JIA Wei LI Lei CAI Yun YING WanTao QIAN XiaoHong 

机构地区：[1]Department of Biology, Energy and Environment, National Institute of Metrology, Beijing 100013, China [2]State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China

出　　处：《Science China(Life Sciences)》2011年第1期25-33,共9页中国科学（生命科学英文版）

基　　金：supported by the National Natural Science Foundation of China (Grant Nos. 30621063, 20635010 and 20735005);the National Key Basic Research Program of China (Grant Nos. 2006CB910801, 2004CB518707 and 2007CB914100);the National High Technology Research and Development Program of China (Grant Nos. 2006AA02A308 and 2008AA02Z309)

摘　　要：The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3-10 and 4-7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5-9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project （HLPP）, the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis （2-DE）. We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple （overlapping） narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.

关 键 词：human liver proteome two-dimensional gel electrophoresis ultra-zoom gel mass spectrometry 

分 类 号：Q51[生物学—生物化学] P457.6[天文地球—大气科学及气象学]