Liquid chromatography electrospray ionization and matrix-assisted laser desorption ionization tandem mass spectrometry for the analysis of lipid raft proteome of monocytes.
Lipid rafts are dynamic assemblies of cholesterol and glycolipid that form detergent-insoluble microdomains within membrane lipid bilayers. Because rafts can be separated by flotation on sucrose gradients, interrogation by mass spectrometry (MS) provides a valuable new insight into lipid raft function. Here we combine liquid chromatography (LC) electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) MS/MS to corroborate and extend our previous description of lipid raft proteomes derived from the monocytic cell line THP-1. Interestingly, LC-ESI and MALDI MS/MS identify largely non-overlapping, and therefore, potentially complementary protein populations. Using the combined approach, we detected 277 proteins compared to 52 proteins obtained with the original gel-based MALDI MS. We confirmed the presence of 47 of the original 52 proteins demonstrating the consistency of the lipid raft preparations. We demonstrated by immunoblotting that Rac 1 and Rac 2, two of the 52 proteins we failed to confirm, were indeed absent from the lipid raft fractions. The majority of new proteins were cytoskeletal proteins and their regulators, proteins implicated in membrane fusion and vesicular trafficking or signaling molecules. Our results therefore, confirm and extend previous evidence indicating lipid rafts of monocytic cells are specialized for cytoskeletal assembly and vesicle trafficking. Of particular interest, we detected SNAP-23, basigin, Glut-4 and pantophysin in lipid rafts. Since these proteins are implicated in both vesicular trafficking and gamete fusion, lipid rafts may play a common role in these processes. It is evident that the combination of LC-ESI and LC-MALDI MS/MS increases the proteome coverage which allows better understanding of the lipid raft function.
Zhang
Nan
N
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Shaw
Andrew R E
AR
Li
Nan
N
Chen
Rui
R
Mak
Allan
A
Hu
Xiuying
X
Young
Nelson
N
Wishart
David
D
Li
Liang
L
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002853:Chromatography, Liquid; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D008970:Molecular Weight; D009000:Monocytes / Q000737:chemistry / Q000166:cytology*; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D020744:rac GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000032:analysis / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
D006160:Guanosine Triphosphate; D010455:Peptides; D020543:Proteome; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein