Investigation And Application Of Two-dimensional Gel Electrophoresis Technique (2-DE) In Proteomic Research

With introduction of immobilized pH gradients (IPGs), problems of pH-gradientinstability and reproducibility of two-dimensional gel electrophoresis(2-DE)havebeen largely overcome, which was become one of the important techniques inproteomic research. However, a series of problems in IPG-Dalt still remains to besolved for the instability of each operated step of 2-DE. In proteomic research, one ofmissions is to separate all target proteins and then identify them. Therefore, 2-DEbecomes the first choice because it is capable of simultaneously separating thousandsof proteins for quantitative comparison at a time. 2-DE with high resolution, high throughput and high reproducibility was priorlyset up and improved in our lab in China. And then a new 2-DE protocol which namedmulti-IPGstrips gels running simultaneously on one sodiumdodecyl sulfate (SDS) gel(MSOG) method was put forward and furthered these properties. Based on thismethod, nineteen proteins showing significant difference in expression were selectedand identified by matrix assisted laser desorption / ionization-time of flight-massspectrometry (MALDI-TOF-MS) and database search when the proteomic profiles ofa human hepatoma revertant CL1and its original cell line SMMC7721 were compared.These proteins have direct or indirect relationship to the phenotypic reversion ofSMMC7721. At the same time, these proteins such as maspin and cathepsin D are ofpotential importance as diagnostic and therapeutic markers for human hepatocellularcarcinoma. It is a milestone to diagnose disease and evaluate curative effect in the medicinecopyright 2004 中科院上海生物化学与细胞生物学研究所博士论文 2双向凝胶电泳技术研究及其在蛋白质组学中的应用 机密by using serum proteomics which was the most remarkable field in the proteomicresearch. In order to directly study important disease and clinical medicine, we payour emphase from cultured cells to serum proteomics. We took mouse as model andexplored the effect of long-term exercise to Cpe fat/fatmice (due to mutation ofcarboxypeptidase E)and wild type mice. The result indicated that the expression levelof a series of serum proteins such as haptoglobin and some kind of kallikreins werechanged by 2-DE / MS. Based on these study, not only 2-DE with high reproducibilityin serum proteomics was set up, but also a series of important proteins related toobesity were discovered. It was helpful to figure out the mechanism of exercise andobesity, at the same time, it established a well approach to find important biomarkers.