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Comparison of EVs isolated from human plasma using different methods

ABSTRACT: Comparison of different EV isolation methods for their proteomic coverage

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Qibin Zhang

LAB HEAD: Qibin Zhang

PROVIDER: PXD058948 | Pride | 2025-06-16

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	Cell_EV_1.raw	Raw
	Cell_EV_2.raw	Raw
	DGUC_1.raw	Raw
	DGUC_2.raw	Raw
	DGUC_3.raw	Raw

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Publications

Comprehensive Comparison of Methods for Isolation of Extracellular Vesicles from Human Plasma.

Suresh Patil Shivprasad PS Zhang Qibin Q

Journal of proteome research 20250512 6

Extracellular vesicles (EVs) are a vital component in cell-cell communication and hold significant potential as biomarkers and therapeutic carriers. Having a reproducible and simple EV isolation method for small volumes of human plasma is essential for biomarker discovery. Although combining multiple methods has been a recent trend in its ability to minimize contamination, it is not ideal for clinical specimens due to the large sample number and small sample volume. This study compared EVs isola ...[more]

PMID: 40356199

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Comparison of EVs isolation methods for miRNAs sequencing

Project description:Background & aims: MicroRNAs (miRNAs) encapsulated in EVs are potential diagnostic and prognostic biomarkers. However, discrepancies on miRNA patterns and their validation are still frequent due to differences in sample origin, EVs isolation, miRNA extraction and sequencing methods. Selecting appropriate EVs isolation methods is therefore a critical step for miRNA-based biomarker discovery. The aim of the present study is to find the most suitable EVs isolation method for miRNAs sequencing adequate for clinical application. Material & Methods EVs were isolated by Size Exclusion Chromatography (SEC), iodixanol gradients (GRAD) and the combination of both (SEC+GRAD), using the same plasma sample, in triplicate isolation assays. Isolated EVs were characterized and RNA was extracted. Three different protocols for miRNA library preparation were compared (NEBNext, NEXTFlex and SMARTer smRNA-seq) and miRNAs encapsulated on EVs were sequenced using NextSeq 500 system (Illumina). Finally, the yield, abundance and diversity of miRNAs using the three different EVs isolation protocols were analyzed and compared between them. Results The majority of lipoproteins, total cholesterol and plasma proteins were removed from the EVs-containing fractions by using SEC, GRAD, and SEC+GRAD. SEC method recovered a larger amount of EVs followed by GRAD and SEC+GRAD, while GRAD and SEC+GRAD yielded the purest vesicles. NEBNext was the library preparation kit that showed the highest reproducibility among replicas, higher number of reads corresponding to miRNAs and more different miRNAs, followed by NEXTFlex and SMARTer smRNA-seq. GRAD method showed the highest reproducibility among replicas, a higher number of reads corresponding to miRNAs and more different miRNAs, followed by SEC and SEC+GRAD methods. Conclusions These results render the GRAD method to isolate EVs as one of the most appropriate to detect miRNAs from Evs.

2023-04-26 | GSE223838 | GEO

Comparison of deep plasma proteome enrichment methods

Project description:Plasma is a complex biological fluid containing extracellular vesicles (EVs), residual platelets, and soluble proteins, all of which can serve as biomarkers for pathological conditions. While conventional plasma proteomics typically identifies hundreds of proteins, recent enrichment strategies have expanded coverage to thousands. It is not clear yet whether these methods enrich preferentially different classes of protein and whether they allow for reliable quantification Here, we compared three common advanced proteomic workflows—Seer Proteograph (nanoparticle-based), MagNET (SCX magnetic beads), and ENRICHplus (PreOmics)—as well as EV enrichment obtained by centrifugation. We aimed to explore the content in soluble proteins, EV cargo, and platelet-derived proteins in platelet-poor plasma (PPP) from healthy donors after the enrichments. Samples were analyzed using timsTOF platforms and quantified with DIA-NN in library-free mode. Functional insights were obtained using Over-Representation Analysis (ORA), Human Protein Atlas annotations, and EV-specific markers. Quantification was evaluated comparing each method to neat plasma using protein coefficient of variation and point-biserial correlation. We quantified an average of ~4500 proteins with EV centrifugation, ~4000 with Seer, ~2800 with ENRICHplus, ~2300 with MagNET, and ~900 with neat plasma. Each method enriched distinct protein signatures: EV markers such as CD81 were predominantly detected in EV preparations; lipoproteins were enriched in ENRICHplus; cytokine and hormone signaling pathways were most evident in Seer. Platelet protein intensity was directly correlated with total protein identifications but did not compromise quantification of low-abundance proteins. Across 50 healthy individuals, Proteograph consistently demonstrated reproducible enrichment and depletion patterns, with notable exceptions for proteins such as FCN3, F11, and FCGBP.

2025-10-17 | PXD063673 | Pride

Comparison of EVs isolation methods for miRNAs sequencing

Project description:Comparison of EVs isolation methods for miRNAs sequencing

| PRJNA928492 | ENA

Isolation and analysis methods of extracellular vesicles (EVs).

Project description:Extracellular vesicles (EVs) have been recognized as an evolving biomarker within the liquid biopsy family. While carrying both host cell proteins and different types of RNAs, EVs are also present in sufficient quantities in biological samples to be tested using many molecular analysis platforms to interrogate their content. However, because EVs in biological samples are comprised of both disease and non-disease related EVs, enrichment is often required to remove potential interferences from the downstream molecular assay. Most benchtop isolation/enrichment methods require > milliliter levels of sample and can cause varying degrees of damage to the EVs. In addition, some of the common EV benchtop isolation methods do not sort the diseased from the non-diseased related EVs. Simultaneously, the detection of the overall concentration and size distribution of the EVs is highly dependent on techniques such as electron microscopy and Nanoparticle Tracking Analysis, which can include unexpected variations and biases as well as complexity in the analysis. This review discusses the importance of EVs as a biomarker secured from a liquid biopsy and covers some of the traditional and non-traditional, including microfluidics and resistive pulse sensing, technologies for EV isolation and detection, respectively.

| S-EPMC8372011 | biostudies-literature

Plasma Cell Type Isolated EVs

Project description:To characterize the EVs produced by blood based cell types

2025-01-31 | GSE253127 | GEO

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

Project description: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.

2024-07-03 | PXD049342 | Pride

DNA extraction methods comparison

Project description:The impact of the DNA extraction methods on the gut bacterial and fungal microbiota community recovery

| PRJEB29363 | ENA

Plasma Cell Type Isolated EVs

Project description:Plasma Cell Type Isolated EVs

| PRJNA1064083 | ENA