(1) Institute of Microbiology, Academy of Science of the Czech Republic, Prague, Czech Republic
(2) Department of Biochemistry, Faculty of Science, Prague, Czech Republic
The aim of our study is to develop a method for complete characterization of proteins associated with lipid rafts of Jurkat T-cell line. Lipid rafts are rich in cholesterol and sphingolipids. Due to the enrichment of cholesterol and sphingolipids in the lipid rafts, they concentrate in low density fractions during the sucrose density gradient ultracentrifugation. Upon cellular activation, signal transduction molecules (src-kinases lck and fyn, CD45 antigen, and others) concentrate in lipid rafts, and initiate polymerization of cytoskeletal proteins leading to the formation of an immune synapse.
While conventional immunochemical staining can reveal only one protein
a single experiment, sensitive and robust mass spectrometric technique provides us with more detailed information on protein content of the lipid raft. We applied a more rigorous purification scheme involving isolation of plasma membranes after hypotonic lysis to exclude the “non-plasmatic” membrane microdomains. Moreover, we used two types of lysis buffers. TNE with 1% NP-40 represents classical extraction used in lipid raft studies. The “non-classical” MES buffer with Na2CO3 allows removal of non-specifically associated proteins. Proteins were identified by two approaches: immunoblot with ECL detection, and microcapillary liquid chromatography in combination with electrospray ionization tandem mass spectrometry (microHPLC-ESI-MS/MS).
By applying these conditions, we have identified a large number of functionally relevant proteins involved in signaling, cytoskeletal association, cellular adhesion and other processes. Western blot analysis has revealed typical GEMs proteins (Lck and CD59) and thus verified the quality of sample preparation. On the other hand mass spectrometry has found not only GEMs markers but also many other molecules involved in cell signaling processes like Ras-related protein, GTP-binding protein, S100-Ca2+ binding proteins, CD45, 4F2, HLA-I, etc.
Financial support: MSM 113100001, AVOZ5020903.