Proteomic study of caveolae and rafts isolated from human endothelial cells

Caveolae and rafts are specialized microdomains of the endothelial cell plasma membrane, which play an important role in signal transduction, transcellular transport, and cholesterol homeostasis. The dynamic protein composition of these subcellular lipid domains has been implicated in a variety of patho-physiological states of the vasculature, and is receiving increased attention. As a result of the membranous composition and abundance of insoluble intrinsic and membrane-associated proteins, determination of the raft/caveolae subproteome composition requires specially adapted methods. In this chapter, we present a straightforward protocol to obtain comprehensive and reliable peptide mixtures from this subproteome by subcellular fractionation and both one-dimensional and two-dimensional gel electrophoresis. These mixtures allow dynamic monitoring of composition and posttranslational modification of the raft/caveola subproteome using peptide mass fingerprinting and direct peptide sequencing tandem mass spectrometry.