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

基于O'Farrell等电点电泳的IPG方法是蛋白质组学中唯一流行的方法。然而，IPG方法有两个严重的弱点，给蛋白质分离步骤带来了困难。这些缺点是IPG方法不能充分分离基本蛋白质，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，可以满足要求。本项目旨在提高极少量蛋白质的分离检测和鉴定能力，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，为了提高RFHR法的分离能力，我们研制了一种严格控制温度的水冷装置。分离能力大大提高，鉴定出的大肠杆菌蛋白基因数量是IPG法的2倍以上，达到568个。这个鉴定的数字对应于CBB染色检测到的700个蛋白点的80%。取消蛋白浓度0D电泳，用0D浓度缓冲液将蛋白浓缩至1D凝胶顶部，再进行1D电泳。通过本次试验，将使RFHR法在今后的改进中更容易实现操作的自动化和便捷化。大鼠肝脏蛋白质组学研究的难点是提高蛋白质的溶解度。我们在0D和1D凝胶中引入了2 M硫代尿素。这种处理可以更有效地溶解较大的膜结合蛋白。人类蛋白质组学研究始于20世纪30年代，并将继续进行下去。

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.