Dissecting the Membrane Proteome of Human Epidermal Melanocytes

摘要

The ability to effectively profile specific cell types purified from human tissues is of fundamental importance since the proteomes isolated from cell lines may not accurately resemble those in situ. Here, we present a robust proteomic approach developed for large-scale profiling of membrane proteins isolated from highly purified melanocyte cell population obtained from human epidermal tissue. This approach relies on BrdU pulse-labeling coupled with flow-sorting-based isolation of targeted cell population. To characterize the membrane proteome of human melanocytes we purified MART-1(+) melanocytes from BrdU pulse-labeled epidermal neonatal skin grafts, established on the backs of nude mice, using flow sorting. Microsomal melanocyte fraction was subsequently isolated by ultracentrifugation and analyzed by 2-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS). 2D-LC-MS/MS analysis resulted in the identification of 31,845 fully tryptic peptides which allowed a total of 4,457 proteins to be identified by at least two unique protein specific peptides.