Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line.
Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.
Pompach
P
P
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. pompach@biomed.cas.cz
Man
P
P
Novák
P
P
Havlícek
V
V
Fiserová
A
A
Bezouska
K
K
D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D007477:Ions; D019169:Jurkat Cells; D013058:Mass Spectrometry / Q000379:methods*; D021962:Membrane Microdomains / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D013223:Statistics as Topic / Q000379:methods; D013395:Sucrose / Q000494:pharmacology
D003902:Detergents; D007477:Ions; D011506:Proteins; D013395:Sucrose