细胞培养条件下稳定同位素标记技术(stable isotope labeling by amino acids in cell culture,SILAC)是一种简单的体内标记策略,是基于质谱的定量蛋白质组学技术中的一个有力工具。SILAC技术不会引起蛋白质互作研究中的假阳性结果,使细胞信号转导动态变化研究成为可能,并且能够直接测量蛋白质含量。SILAC技术已经应用于一系列的生物学研究中,近来已被成功应用到整体动物水平的定量蛋白质组研究。本文综述了SILAC技术的基本原理、优点和应用进展。
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.
MI WeiLIU XinJIA WeiLI LeiCAI YunYING WanTaoQIAN XiaoHong
