Transcriptome-based identification of candidate membrane proteins
Evans EJ, Hene L, Vuong M, Abidi HS, Davis SJ. (2009), Methods Mol Biol. 528, 37-56
A full understanding of leukocyte responses to external stimuli requires knowledge of the full complement of proteins found on their surfaces. Systematic examination of the mammalian cell surfaces at the protein level is hampered by technical difficulties associated with proteomic analysis of so many membrane proteins and the large amounts of starting material required. The use of transcriptomic analyses avoids challenges associated with protein stability and separation and enables the inclusion of an amplification step; thus allowing the use of cell numbers applicable to the study of sub populations of, for example, primary lymphocytes. Here we present a transcriptomic methodology based on Serial Analysis of Gene Expression (SAGE) to recover an essentially complete and quantitative profile of mRNA species in a particular cell. We discuss how, using bioinformatic tools accessible to standard desktop computers, plasma membrane proteins can be identified in silico, from this list. While we describe the use of this approach to characterise the cell surface protein complement of a resting CD8(+) T-cell clone, it is theoretically applicable to any cell surface, where a suitable pure population of cells is available.