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Deep proteomic analysis of circulating large/small extracellular vesicle enriched fraction based on data-independent acquisition mass spectrometry

ABSTRACT: Proteins in the plasma/serum mirror an individual’s physiology. Circulating extracellular vesicle (EVs) proteins constitute a large portion of the plasma/serum proteome. Thus, deep and unbiased proteomic analysis of circulating plasma/serum extracellular vesicles holds promise for discovering disease biomarkers as well as revealing disease mechanisms. We established a workflow for simple, deep, and reproducible proteome analysis of both serum large and small EVs enriched fractions by ultracentrifugation plus 4D-DIA-MS. In our cohort study of obstetric antiphospholipid syndrome (OAPS), 4270 and 3328 proteins were identified from large and small EVs enriched fractions respectively. Both of them revealed known or new pathways related to OAPS. Increased levels of von Willebrand factor (VWF) and insulin receptor (INSR) were identified biomarker features, which sheds light on hypercoagulability and insulin signaling in disease progression. Our workflow for deep quantitative proteome of EV enriched fraction combined with targeted validations will significantly promote our understanding of plasma/serum-based disease mechanisms and generate new biomarkers.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yang Chen

PROVIDER: PXD043290 | iProX | Mon Jun 26 00:00:00 BST 2023

REPOSITORIES: iProX

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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.

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Deep proteomic analysis of circulating large/small extracellular vesicle enriched fraction based on data-independent acquisition mass spectrometry

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