(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
in
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.