Dataset Information

Quick steps to get glioma extracellular vesicles surface membrane proteins mapping based on tryspin/Lysin C digestion followed by Amicon and shot gun proteomic: identification of ghost protein

ABSTRACT: Extracellular vesicles (EVs) are known to mediate intercellular communication and regulate a diverse range of crucial biological processes. Novel therapeutic strategies have emerged based on these EVs as biological nanoparticles. EVs is as a generic term for all secreted vesicles released including at least ectosomes, microvesicles, apoptotic bodies and exosomes. In order to determine if it is possible to get access to the protein present at the surface of the EVs, we developed first a quickstep strategy. We performed a Trypsin/Lys C digestion treatment for EVs pellet, followed by an Amicon filtration. This procedure sep-arates the EVs from peptides of protein aggregates and external part of EVs membrane proteins. After separation step, all the fractions have been subjected of proteomic analyses. Comparison between T-0h and T-6h, Trypsin/Lysin C digestion revealed an evolution of the surface EVs proteins. Some surface proteins have been demasked after 6h enzymatic digestion like CD81, CD82 ,Ust, Vcan, Lamp 1,rab43, Cspg4, Annexin A2, Synthenin 1,VSP37b. These results established an evolu-tion in time course of the nature of the surface proteins in glioma tumor.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Vesicle, Cell Culture

SUBMITTER: Soulaimane Aboulouard

LAB HEAD: Michel Salzet

PROVIDER: PXD016944 | Pride | 2021-02-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	AltProt-AdriannaArticle-Exosome.pdResult	Other
	RAW-files.zip	Other
	Reading-samples.pptx	Other
	experimentalDesignTemplate.txt	Txt
	modificationSpecificPeptides.txt	Txt

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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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Quick steps to get glioma extracellular vesicles surface membrane proteins mapping based on tryspin/Lysin C digestion followed by Amicon and shot gun proteomic: identification of ghost protein

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