Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization

Abstract

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.

Shed Membrane Particles from Preeclamptic Women Generate Vascular Wall Inflammation and Blunt Vascular Contractility

“…This could be related to the different MP compositions as functions of their origin, as shown in the proteomic analyses from platelet and leukocytic MPs. 35,36 PrMPs from platelets induced vasodilator products, and this property might protect against vasoconstrictor factors generated by leukocyte PrMPs.…”

Circulating Microparticles

“…The pattern of proteins found on MPs (which differs between vesiculation stimuli), as well as the level of oxidized lipids, are likely to influence their effects on target cells. 23,24 In addition, in vitro concentrations of MPs should be consistent with those found in vivo for MPs of the same cellular origin.…”

“…Proteomic analyses have revealed that the spectrum of proteins found in MPs released in vitro from cultured cells is influenced in part by the type of stimulus used to trigger cell vesiculation. 24 This different pattern of protein expression may be helpful in distinguishing within a subpopulation of circulating MPs those released after apoptotic stimuli from MPs resulting of cell activation. 25 …”

Microparticles as Biomarkers of Vascular Dysfunction in Metabolic Syndrome and its Individual Components

“…Molecular profiling approaches using proteomic techniques, performed in order to determine the protein composition of microparticles, have shown that protein composition depends not only on cell type, but also on the conditions of cell stimulation [9,10]. Furthermore, differential lipid composition of microparticles may vary depending on the lipidic environment surrounding cells; thus it is possible that microparticles from patients with metabolic pathologies associated with lipid alterations may have different lipid composition which may account for different functional effects of microparticles on target cells.…”