Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization.
Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.
Miguet
Laurent
L
Laboratoire de Spectrométrie de Masse Bio-Organique, Université Louis Pasteur, CNRS UMR 7512, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France.
Pacaud
Karine
K
Felden
Claire
C
Hugel
Bénédicte
B
Martinez
M Carmen
MC
Freyssinet
Jean-Marie
JM
Herbrecht
Raoul
R
Potier
Noelle
N
van Dorsselaer
Alain
A
Mauvieux
Laurent
L
D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D016393:Lymphoma, B-Cell / Q000378:metabolism* / Q000473:pathology; D036103:Nanotechnology; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D020543:Proteome