One protein/cell proteomics by the high power RFHR-2D-PAGE

基于O‘Farrell等电点电泳法的IPG方法在蛋白质组学领域独树一帜。然而，IPG方法有两个严重的缺点，给蛋白质分离步骤带来了困难。这些缺点是IPG法不能充分分离碱性蛋白质，而且这种方法使蛋白质在2D凝胶上分裂成大约10个点。为了克服这些缺点，需要一种新的方法，它具有不同于IPG方法的新的分离原理。RFHR 2D PAGE不采用等电点电泳法，可以满足要求。本项目的目的是提高对极少量蛋白质的检测和鉴定的分离能力，并利用改进的RFHR方法对大肠杆菌和真核生物进行蛋白质组学研究。首先，在提高RFHR分离能力的基础上，我们研制了严格控制温度的水冷装置。分离能力大大提高，我们发现了568个基因的大肠杆菌蛋白大于两倍的IPG方法。这个鉴定的数字与CBB染色检测到的700个蛋白质点中的80%相对应。取消了蛋白质浓度的0D电泳法，在1D电泳法之前，使用0D浓度缓冲液将蛋白质浓缩到1D凝胶的顶部。通过这次试验，未来RFHR方法的改进将变得更加自动化和方便。第二，真核生物蛋白质组学研究大鼠肝脏蛋白质组学的一个难点是提高蛋白质的溶解度。我们在0D和1D凝胶中引入了2M硫脲。这种处理更有效地溶解了更大的和膜结合的蛋白质。我们从第三年就开始了人类蛋白质组学，并将从现在开始继续下去。

The IPG method based on O'Farrell's iso-electric point electrophoresis is exclusively popular on proteomics. The IPG method, however, has two serious weak points, and has brought about a difficulty to the protein separation step. These weak points are that the IPG method can not separate basic proteins sufficiently, and that this method makes proteins split on 2D gels to about ten spots. To conquer these weak points, a new method is necessary, which has a new separation principle different from that of the IPG method. The RFHR 2D PAGE can satisfy the request, because it does not adopt the iso-electric point electrophoresis. The aim of this project is to improve the separation ability for detection and identification of extremely small amount proteins, and to carry out proteomics for E.coli and eukaryotes by the improved RFHR method. First, on the increase of separation ability of the RFHR method We developed a water- cooled apparatus to control temperature strictly. The separation ability increased greatly, and we identified 568 genes for E.coli proteins larger than two times as much as those by the IPG method. This identified number corresponds to 80% of 700 protein spots detected with CBB staining. The 0D electrophoresis for protein concentration was abolished, and the proteins were concentrated into the top of the 1D gels using the 0D concentration buffers, prior to the 1D electrophoresis. By this trial, future improvement of the RFHR method to automatic and convenient operations will become more easy. Secondly, on proteomics of eukaryotesA difficult point on rat liver proteomics is to increase solubility of proteins. We introduced 2 M thio-urea to the 0D and 1D gels. This treatment solubilized larger and membrane bound proteins more effectively. We began human proteomics from the 3^<rd> year, and will continue it from now on.