Mass spectrometric characterization of proteins extracted from Jurkat T cell detergent-resistant membrane domains

Plasma membranes of most cell types are thought to contain microdomains com monly referred to as lipid rafts, biochemically distinct from bulk plasma m embrane and apparently enriched for proteins involved in signal transductio n. In T cells, it is believed that lipid rafts aggregate at the site of T c ell receptor engagement and act as foci for initiation of the signaling pro cess. In order to gain insight into the possible functioning of lipid rafts , we applied microcapillary liquid chromatography-electrospray ionization-t andem mass spectrometry (mu LC-ESI-MS/MS) methodologies to the identificati on of proteins which co-purified with lipid rafts. Following isolation of l ipid rafts as Triton-insoluble, low-density membrane fractions from Jurkat T cells, tryptic digests were generated of electrophoretically-resolved, in dividual protein bands. Alternatively, avidin-affinity purification was use d to isolate cysteine-containing peptides from total tryptic digests of uns eparated lipid raft proteins following protein labeling with a cysteine-spe cific biotinylation reagent. In both cases, protein identifications were ma de by comparison of tandem mass spectra, generated by mu LC-ESI-MS/MS, with both protein and DNA sequence databases using Sequest software. Proteins i dentified essentially fell into two groups: cytoskeletal proteins and prote ins involved in signal transduction. These findings are discussed in light of the current understanding of both lipid-raft biology and signal transduc tion.