Optimization Of The Two-dimensional Electrophoresis Protocol And Differential Proteomics Analysis Of Human Infiltrating Ductal Carcinoma

Breast cancer is one of the most frequent female tumors. The incidence has an increasing trend all over the world. The epidemiological study on the breast cancer etiology suggested that the occurring may be related with some physiological factors, genetic factors and other environmentally hazardous factors.Recent studies on breast cancer carcinogenesis have greatly driven into the molecular mechanism. Some breast cancer susceptible and related genes have been found. However, all of the phenotypes are hardly interpreted at gene level. This has led molecular biological research on carcinogenesis entered the functional genomics era of which proteomics is considerated to be an effective tool.It was less than 15 years since the term"proteomics"was produced. The breast cancer related database has not yet been completed. The aim of this study is to compare the difference between normal breast tissue and breast solid tumor tissue and to explore the differentially expressed proteins, so as to elucidate the proteomic change in the procedure of the breast cancer occurring and development.This study identifies the differentially expressed proteins by the means of extraction of the whole-cell proteins, protein purification, optimization of 2-DE protocols, 2-DE gel imaging, and gel image analysis.The on-gel differentially expressed protein spots were then identified by MELDI-TOF-TOF MS.We first optimized the popularly used 2-DE protocols to ensure the suitable one for this study. This included the storage of the breast tissue biopsy, the crash down of the tissue cells, the effects of the lysis solution and the effects of differential precipitation. After the comparison, we chose LSⅢ, and acetone/ methanol precipitation for the preparation of the entire breast tissue protein. In the IEF step, we chose the process: pH4-7,18cm IPG gel, 46000Vh,12h30min, silver staining, 280μg, with which could satisfactory 2-DE gel images were achived. After the analysis using the ImageMaster 2D software and Adobe Photoshop software, we found three differentially expressed protein spots all over the 5 breast cancer samples. They were identified by MELDI-TOF or MELDI-TOF-TOF mass spectrum to be respectively immunoglobulin lambda chain variable region, collagen alpha-3(VI) chain precursor and S100 calcium binding protein A11. The change of immunoglobulin lambda chain variable region may be caused by the change of the immunity system of the body. Collagen alpha-3(VI) chain precursor may be associated with the formation of the IDCA solid tumor process. S100 calcium binding protein A11 was believed to related to the invasive process of IDCA.An effective proteomic method for breast cancer tissue was successfully established in this study. The three proteins found in this study may like to be potential biomarker candidates of the infiltrating ductal carcinoma, which warrants futher investigation.