Dataset Information

Profiling of Human Circulating Plasma Microparticle Associated Proteins by DDA and DIA

ABSTRACT: Cells of the vascular system release spherical vesicles, called microparticles (MP), in the size range of 0.1-1μm induced by a variety of stress factors resulting in variable MP concentrations between health and disease. Furthermore, MP have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today’s most used analytical technology for MP characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past. We propose the use of nano-liquid chromatography coupled to two-stage mass spectrometry as a non-biased tool for quantitative MP proteome analysis. Using aliquots of 250 μL platelet-free plasma (PFP) from one individual donor, we achieved excellent inter-assay CV’s of 2.7 ± 1.7% (mean ± 1 S.D.) on individual peptide intensities across 27 data-dependent nanoLC-MS/MS runs performed over a period of 3.5 months. With quantitative proteomics, we show that MP composition between twelve volunteers were remarkably stable. MP protein composition is clearly distinguishable from whole cell lysates and we propose that this trait should be used as a quality criterion of MP purity. Furthermore, MP were damaged by freezing PFP. The damage was articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition (cell markers) was not affected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood Plasma

DISEASE(S): Disease Free

SUBMITTER: Manfred Heller

LAB HEAD: Manfred Heller

PROVIDER: PXD003935 | Pride | 2016-10-18

REPOSITORIES: Pride

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Publications

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins.

Braga-Lagache Sophie S Buchs Natasha N Iacovache Mircea-Ioan MI Zuber Benoît B Jackson Christopher Benjamin CB Heller Manfred M

Molecular & cellular proteomics : MCP 20161012 12

Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1 μm induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might ha ...[more]

PMID: 27738094

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Project description:Cerebral malaria (CM) is a severe complication of a Plasmodium falciparum infection and is still responsible for a high number of deaths and long term disabilities in children in sub-Saharan Africa. The pathogenesis of this syndrome remains incompletely understood but a number of mechanisms and effectors have been proposed, including plasma microparticles (MP). These submicron vesicles released by potentially all cells carry proteins and nucleic acids derived from the mother cell. In CM, their numbers are increased in patients’ circulation and, in the mouse model, they can be localised within inflamed vessels, suggesting their involvement in vascular damage. In the present work we define, for the first time, the protein cargo of MP during experimental cerebral malaria (ECM) with the overarching hypothesis that characterising this content could help in better understand CM pathogenesis. Using qualitative and quantitative high-throughput proteomics we compared MP proteins from non-infected mice and mice infected with P. berghei ANKA at two different stages of infection, i.e., pre-clinical phase and full blown syndrome. Overall, we identified 368 proteins, 60 of which were differentially expressed, as determined by quantitative comparison using the TMT® isobaric labelling. The analysis with Ingenuity® IPA highlighted important networks significantly represented among the proteins over-expressed (n=42) or identified only (n=21) in ECM, comprising mechanisms already proposed to be involved in CM, such as endothelial activation. Two proteins of interest, carbonic anhydrase I (CA-I) and S100A8, were verified by western blot on newly collected MP samples from DBA/1 (n=16) and C57BL/6 (n=8) mice, and confirmed to be associated with ECM MP. These results demonstrate that MP protein cargo represents a novel ECM pathogenic trait to consider in the understanding of CM pathogenesis. The identification of the cellular source of MP containing proteins associated to the disease state will contribute to further understanding their involvement in CM.

Dataset Information

Profiling of Human Circulating Plasma Microparticle Associated Proteins by DDA and DIA

Publications

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins.

Similar Datasets

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