Dataset Information

Evaluation of depletion of abundant plasma proteins for proteomics characterization of EVs

ABSTRACT: In this study, we aimed to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC-MS/MS by improving the proteome coverage. Plasma depletion was performed using single-use spin columns prior to SEC-based EV isolation. Afterwards, EVs derived from undepleted and depleted plasma were characterized by mass spectrometry (MS)-based proteomics using a data-independent acquisition (DIA) approach.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Sandrine Reymond

LAB HEAD: Jean-Charles Sanchez

PROVIDER: PXD039240 | Pride | 2023-07-20

REPOSITORIES: Pride

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Publications

Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls.

Reymond Sandrine S Gruaz Lyssia L Sanchez Jean-Charles JC

Analytical and bioanalytical chemistry 20230418 16

Blood extracellular vesicles (EVs) play essential roles in cell-cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could impro ...[more]

PMID: 37069444

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

Dataset Information

Evaluation of depletion of abundant plasma proteins for proteomics characterization of EVs

Publications

Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls.

Similar Datasets

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