Mass Spectrometric Characterization of Proteins Extracted from Jurkat T Cell Detergent-Resistant Membrane Domains

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Abstract
Plasma membranes of most cell types are thought to contain microdomains commonly referred to as lipid rafts, biochemically distinct from bulk plasma membrane and apparently enriched for proteins involved in signal transduction. In T cells, it is believed that lipid rafts aggregate at the site of T cell receptor engagement and act as foci for initiation of the signaling process. In order to gain insight into the possible functioning of lipid rafts, we applied microcapillary liquid chromatography-electrospray ionization-tandem mass spectrometry (μLC-ESI-MS/MS) methodologies to the identification of proteins which co-purified with lipid rafts. Following isolation of lipid rafts as Triton-insoluble, low-density membrane fractions from Jurkat T cells, tryptic digests were generated of electrophoretically-resolved, individual protein bands. Alternatively, avidin-affinity purification was used to isolate cysteine-containing peptides from total tryptic digests of unseparated lipid raft proteins following protein labeling with a cysteine-specific biotinylation reagent. In both cases, protein identifications were made by comparison of tandem mass spectra, generated by μLC-ESI-MS/MS, with both protein and DNA sequence databases using Sequest software. Proteins identified essentially fell into two groups: cytoskeletal proteins and proteins involved in signal transduction. These findings are discussed in light of the current understanding of both lipid-raft biology and signal transduction.