Dataset Information

Comparison of three methods for isolating human plasma EVs by LC-MSMS

ABSTRACT: The lack of standardized protocols for isolating extracellular vesicles (EVs), particularly from biobank-stored plasma, poses a substantial limitation for the study of biomarkers. Combining current isolation methods could enhance the specificity and purity of EVs isolation for their further use in diagnosis and personalized medicine. In this study, plasma samples from healthy individuals were subjected to extracellular vesicle isolation using ultracentrifugation (UC), size exclusion chromatography (SEC), and a combined approach of SEC and UC (SEC+UC). Characterization of the isolated EVs with NTA and TEM indicated that all isolation methods successfully isolated EVs, being not altered due to the long-term storage. Also, the western blot analysis confirmed the presence of exosome markers in all isolates but showed a higher expression of albumin in EVs-UC, suggesting potential plasma protein contamination. Proteomic analysis of samples from different isolation methods identified 542 proteins, with SEC+UC yielding the most complex proteome at 364 proteins. GO analysis of cellular component revealed differences in terms related to EVs and plasma, where SEC+UC proteins had the highest proportion of exosomal proteins. As a result of further study of the unique proteins detected in each isolation method, it was revealed that the SEC+UC method detected 181 unique proteins, demonstrating its superiority in the detection of low plasma concentration proteins. These findings support the robustness of the SEC+UC approach for EV isolation and proteomic studies due to its high efficiency and purity in isolating EVs. This study emphasizes the efficacy of the SEC+UC approach in obtaining highly pure and diverse EV populations suitable for comprehensive proteomic analysis with application to the discovery of biomarkers from biobank-stored plasma.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Ana Torres

LAB HEAD: Eugenia Carrillo

PROVIDER: PXD049342 | Pride | 2024-07-03

REPOSITORIES: Pride

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Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored Plasma Samples.

Torres Ana A Bernardo Lorena L Sánchez Carmen C Morato Esperanza E Solana Jose Carlos JC Carrillo Eugenia E

Biological procedures online 20240619 1

<h4>Background</h4>The lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes.<h4>Results</h4>EVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusi ...[more]

PMID: 38898416

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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.

Project description:Extracellular vesicles (EVs) derived from pleural effusion (PE) is emerging as disease biomarkers. However, the methods for isolation of EVs from PE (pEVs) were rarely studied. In our study, three methods for isolating pEVs of lung cancer patients were compared, including ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC) and a combination of UC and density gradient ultracentrifugation (UC-DGU). The subpopulation of pEVs was identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB) and nanoflow cytometry (nFCM). Additionally, the proteomic landscape of pEVs was analyzed by Label-free proteomics. The results showed that compared with UC and UC-DGU, the UC-SEC method separated pEVs with the highest purity. In the proteomic analysis, on average, 1595 proteins were identified in the pEVs isolated by UC-SEC, much more than pEVs isolated by UC (1222) or UC-DGU (807). Furthermore, approximately 90% of identified proteins in each method were found in the EVs public database ExoCarta. Consistent with this, GO annotation indicated that the core proteins identified in each method were mainly enriched in “extracellular exosome”. Many of the top 100 proteins with high expression in each method were suggested as protein markers to validate the presence of EVs in the MISEV2018 guidelines. In addition, combined with lung tissue-specific proteins and vesicular membrane proteins, we screened out and validated several novel protein markers (CD11C, HLA DPA1 and HLA DRB1), which were enriched in pEVs rather than in plasma EVs. In conclusion, our study shows that the method of UC-SEC could significantly improve the purity of EVs and the performance of mass spectrometry-based proteomic profiling in analyzing pEVs. The exosomal proteins CD11C, HLA DPA1 and HLA DRB1 may act as potential markers of pEVs. The proteomic analysis of pEVs provides important information and new ideas for studying diseases complicated with PE.

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