For quite some time, the standard approach for comprehensive profiling of protein expression in tissues an cell populations has been two-dimensional (2-D) gel electrophoresis and silver staining, coupled more recently with mass spectrometry for protein identification. This approach is currently in use in the PI's laboratory, fo NCI funded projects whose objectives are the molecular analysis of tumors to devise novel classification schemes of cancer, and the identification of novel protein markers for early cancer diagnosis. The standard 2- approach generally allows detection of 1000-2000 protein isoforms in a tissue sample, which is far short of the number of protein isoforms contained in a cell or tissue type. In particular, the 2-D analysis of whole cell lysates yields few proteins from cellular compartments such as membrane proteins. The tagging of proteins b, biotinylation has been extensively utilized for the selective capture and analysis of individual proteins an. antigens and has the potential for providing a substantial increment in sensitivity for the analysis of whole subsets of proteins. Preliminary data obtained by the applicant group demonstrate a remarkable increase in sensitivity and in the yield of low abundance membrane proteins using this approach. The objectives of this application are to develop procedures for the systematic separation, quantitative analysis, identification anc databasing of biotinylated membrane proteins from cells and tissues, that take advantage of the increased sensitivity of this approach. The goals of the R21 phase are to evaluate the utility of the membrane. biotinylation procedure by demonstrating that biotinylated proteins indeed represent membrane proteins; anc that their 2-D patterns are informative with respect to cellular lineage and differentiation state; and that it if feasible to apply this procedure for the selective biotinylation of membranes in intact tissue. Achievement o these milestones will lead in the R33 phase to further optimization of biotinylation procedures; to the development of a database of identified membrane proteins and of their expression patterns, to serve as ; resource for other investigators; and to the development of liquid chromatography based procedures for the separation of biotinylated proteins coupled with their mass spectrometry based identification.
