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Cerebrospinal fluid-derived extracellular vesicles: a proteomic and transcriptomics comparative analysis of enrichment protocols

ABSTRACT: Proteomic and transcriptomic analyses of cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) offer unique insights into molecular changes associated with central nervous system (CNS) diseases and may result in biomarker identification. No gold standard method to enrich EVs from CSF is established and head-to-head comparisons of outputs of different protocols s’ outputs are scarce. Using a large pool of CSF, we characterized the EV preparations resulting from four enrichment protocols and compared them in terms of yield and purity. We found that particles enriched by ultracentrifugation (UC) or a combination of ultrafiltration and size exclusion chromatography (UF-SEC) bared exhibited the typical morphological and biochemical characteristics of small EVs and were highly enriched in proteins and polyadenylated transcripts associated with EV-related biological processes. UF-SEC preparations had higher particle yields while more proteins were identified in UC preparations. Approximately 40% of the EV preparations’ proteome was not identified in unenriched CSF, among which a core proteome of 45 proteins identified in 30 EV preparations from independent experiments which may serve as CSF-derived EV markers. Enrichment scores to protein contaminants albumin and apolipoprotein E were higher in UF-SEC preparations. In conclusion, all protocols analyzed here resulted in enrichment of particles with small EVs characteristics, with EV enrichments from UF-SEC resulting in highest yield and purity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300627 | GEO | 2025/08/20

REPOSITORIES: GEO

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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: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) 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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Cerebrospinal fluid-derived extracellular vesicles: a proteomic and transcriptomics comparative analysis of enrichment protocols

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