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, apparently enriched for proteins involved in signal transduction. In T cells, it is believed that lipid rafts, aggregate at the site of T cel l receptor engagement and act as foci for initiation of the signaling proce ss. In order to gain insight into the possible functioning of lipid rafts, we applied microcapillary liquid chromatography electrospray ionization tan dem mass spectrometry (mu LC-ESI-MS/MS) methodologies to the identification of proteins which copurified with lipid rafts. Following isolation of lipi d rafts as Triton-insoluble, low-density membrane fractions from Jurkat T c ells, tryptic digests were generated of individual protein bands resolved e lectrophoretically. Alternatively, cysteine-containing peptides were isolat ed from total tryptic digests of unseparated lipid raft proteins following labeling with a oysteine-specific biotinylation reagent and avidin affinity purification. In both, cases, protein identifications were made by compari son of tandem MS spectra generated by mu LC-ESI-MS/MS to both protein and D NA sequence databases using Sequest software. Proteins identified essential ly fell into two groups: cytoskeletal proteins, and proteins involved in si gnal transduction. These findings are discussed in the light of the current understanding of both lipid raft biology and signal transduction.