Membrane protein analysis of human breast cancer cell line MCF-7 by different membrane washing methods.

Membrane and membrane-associated proteins are rich in known or potential pharmaceutical drug targets for carcinogenesis. In order to systemically analyze membrane proteins of human breast cancer, we isolated membrane from MCF-7 cells by sequential extraction by washing with three different buffers, namely, phosphate buffer (5 mM, pH 8.0), Tris (40 mM, pH 9.5), and sodium carbonate (100 mM pH 11). The extracted proteins were separated by two-dimensional gel electrophoresis (2-DE) using cup-loading and were then analyzed by peptide mass fingerprinting (PMF). A total of 137 spots from the gels of the three procedures were successfully identified. They corresponded to 79 distinct proteins. Among them, 22 exclusive proteins belonging to each washing procedure were also found, including P-glycoprotein, endoplasmin, Stress-70 protein, ADAM 10, protein disulfide isomerase, and glutamate receptor. These results indicate phosphate buffer to be the most beneficial for enrichment of peripheral membrane proteins, and sodium carbonate is beneficial for the presentation of integral membrane proteins but usually with poor resolution. The reference maps and identified proteins will serve as a basis for the further investigation of breast cancer, especially the proteomic comparison among different cell types of breast cancer, or among the different stages in the drug interfering process of the MCF-7 cell line. Copyright (c) 2008 John Wiley & Sons, Ltd.