Dataset Information

Multimode Chromatography-based Techniques for High Purity Isolation of Extracellular Vesicles from Human Blood Plasma

ABSTRACT: Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation-based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar densities of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography-based strategies. On average, we identified 716±68 proteins and 1054±35 proteins in EV isolates from 100 µL of plasma using multimode chromatography- and ultracentrifugation-based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Alan Zimmerman

LAB HEAD: Alexander Ivanov

PROVIDER: PXD043956 | Pride | 2025-08-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	AZ_20220531_CaptoCore_SEC_2_Rep_1.msf	Msf
	AZ_20220531_CaptoCore_SEC_2_Rep_1.raw	Raw
	AZ_20220531_CaptoCore_SEC_2_Rep_2.raw	Raw
	AZ_20220531_CaptoCore_SEC_2_Rep_3.raw	Raw
	AZ_20220531_CaptoCore_SEC_3_Rep_1.raw	Raw

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Publications

Correction to <i>Journal of Extracellular Biology</i> Articles.

Journal of extracellular biology 20250724 7

[This corrects the article DOI: 10.1002/jex2.70051.]. ...[more]

PMID: 40718630

Similar Datasets

Project description:Growing evidence supports the importance of extracellular vesicle (EV) as mediators of communication in pathological processes, including those underlying respiratory disease. However, establishing methods for isolating and characterizing EVs remains challenging, particularly for respiratory samples. This study set out to address this challenge by comparing different EV isolation methods and evaluating their impacts on EV yield, markers of purity, and proteomic signatures, utilizing equine/horse bronchoalveolar lavage samples. Horses are an excellent translational animal model for respiratory studies due to similarities with human immune responses, shared environmental exposures, and naturally occurring respiratory diseases including asthma. Further, horses are long-lived large animals that allow for longitudinal sample collection, and provide large sample volume and cell yield, which are particularly useful since EV research is commonly limited by low sample yields. Here, EVs were isolated from horse bronchoalveolar lavage fluid (BALF) using four different methods (ultracentrifugation, microcentrifugation, and two sizes of size exclusion chromatography columns) and characterized by measuring particle counts, EV purity, total protein yield, and proteomic cargo, with a specific focus on vesicle surface marker expression potentially informing cell type of origin. We found that size exclusion chromatography yielded the highest particle counts, greatest EV purity markers and elevated vesicle surface marker expression. Overall proteomic profiles differed across isolation methods, with size exclusion chromatography clustering separately from centrifugation. Taken together, our results demonstrate that different isolation methods impact characteristics of EVs, notably that size exclusion chromatography, compared to centrifugation methods, resulted in higher EV purity and better characterized proteomic diversity, including information on EV cell of origin. This is the first study to characterize proteomic profiles of EVs following different isolation methods using equine BALF. The results of this study will pave the way for future studies using equine samples as a model to characterize human respiratory tract EVs.

Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Project description:Extracellular vesicles (EVs) exert important functions in the extracellular space and in cell-to-cell communication. Ultracentrifugation, the most frequently used EV isolation technique, requires specialized equipment and provides relatively low purity and yield. A more recently applied and easily accessible EV isolation technique is size exclusion chromatography (SEC). Here, we hypothesized that EVs isolated from dilute cell culture media by concentration on a 10 kDa filter followed by SEC are suitable for proteomic and functional studies. We also hypothesized that the protein-rich SEC fractions are suitable controls to assess biological effects of the non-EV associated secretome. Cell-depleted medium from BEAS-2B bronchial epithelial cells (60 ml) was 0.22 µm filtered, concentrated to 0.5 ml by 10 kDa UF and run over a sepharose CL-4B column. Fractions were assessed for protein and EV content. EV-rich and protein-rich fractions were concentrated by 10 kDa UF. EV concentrates were characterized by tuneable resistive pulse sensing, cryo transmission electron microscopy and nanoLC-MS/MS. Finally, we assessed the effect of EVs or free secreted protein from unexposed cells or cells exposed to 1% (v/v) cigarette smoke extract (CSE) on IL-8 release by naïve BEAS-2B cells or on adhesion of THP-1 monocytes to endothelial cells. UF concentrated the EVs with approximately 100% recovery. Most of EVs eluted in SEC fractions 6-11, while protein became detectable at fraction 12. The final EV recovery was 40 ± 4%, as compared to 27 ± 8% by ultracentrifugation. Mass Spectrometry of the EV fractions identified 388 different proteins, including various EV markers. Only the protein, but not the EV fractions from media of CSE-exposed cells induced IL-8 release by naïve bronchial epithelial cells, whereas only EVs, but not the protein fractions induced THP-1 adhesion to endothelial cells. Thus, UF followed by SEC provides EV isolates with sufficient yield and purity for proteomic studies and allows directly comparing the functional effects of EVs and the non-EV associated secretome.

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

Multimode Chromatography-based Techniques for High Purity Isolation of Extracellular Vesicles from Human Blood Plasma

Dataset's files

Publications

Correction to <i>Journal of Extracellular Biology</i> Articles.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Multimode Chromatography-based Techniques for High Purity Isolation of Extracellular Vesicles from Human Blood Plasma

Dataset's files

Publications

Correction to &lt;i&gt;Journal of Extracellular Biology&lt;/i&gt; Articles.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Correction to <i>Journal of Extracellular Biology</i> Articles.