Dataset Information

CD9-HPLC-IAC Isolation of Serum Extracellular Vesicles for Proteomic Analysis

ABSTRACT: We developed a column-based CD9 antibody-immobilized HPLC immunoaffinity (CD9-HPLC-IAC) technology for EV isolation from a microliter-scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 μL of serum in 30 min with a yield of 8.0×109 EVs. Proteomic analysis showed that the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101 were all identified in EVs. Statistical analysis of EV protein content showed that the top 10 serum proteins in EVs was significantly decreased by using the CD9-HPLC-IAC method compared to the ultracentrifugation (UC) (p=0.001) and size exclusion chromatography (SEC) (p=0.009), and apolipoproteins significantly reduced 4.8-fold compared to the SEC method (p<0.001). The result demonstrated the potential of the CD9-HPLC-IAC method for efficient isolation and proteomic characterization of EVs from a micro-scale volume of serum.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jianhui Zhu

LAB HEAD: David M. Lubman

PROVIDER: PXD027025 | Pride | 2022-10-14

REPOSITORIES: Pride

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Publications

Column-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles.

Zhu Jianhui J Zhang Jie J Ji Xiaohui X Tan Zhijing Z Lubman David M DM

Journal of proteome research 20210902 10

Serum-derived extracellular vesicles (EVs) are a promising source of biomarkers; however, major challenges in EV separation and proteomic profiling remain for isolating EVs from a small amount, that is, on the microliter scale, of human serum while minimizing the contamination of blood proteins and lipoprotein particles coeluting in EV preparations. Herein we have developed a column-based CD9-antibody-immobilized high-performance liquid chromatography immunoaffinity chromatography(CD9-HPLC-IAC) ...[more]

PMID: 34473505

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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) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. We have previously demonstrated that ultracentrifuged serum is depleted of EVs and, when used in cell culture media, is associated with declines in growth and viability of numerous cultured cell types. Although EVs had been reported to enhance or interfere with HIV-1 infection, depending on the setting, the effects of EVs on HIV-1 production and infectivity of released virions were unknown. In this study, we examined the effects of EV-depleted serum on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Cell culture media were prepared with EV-replete fetal bovine serum (FBS) or serum depleted of EVs via ultracentrifugation or a proprietary method (ThermoFisher/Gibco). T-cell and myeloid-lineage cell lines, including ACH-2 and U1 HIV-1 latency models, and primary cells were grown in 10% FBS-based culture media. Cell counts, viability, and proliferation were assessed throughout. HIV-1 production and infectivity were measured by p24 ELISA and luciferase reporter cell lines, respectively. Flow cytometry, Seahorse assays, and miRNA and mRNA expression arrays were done to assess cellular responses to EV-depleted conditions. Significant increases in HIV-1 production were observed in EV-depleted conditions, along with, in some cases, morphology changes and decreased cell viability. Add-back of pelleted EVs reduced HIV-1 production almost to baseline. Primary cells appeared to be less susceptible to EV depletion. ACH-2 and U1 latency models also produced more HIV-1 under EV-depleted conditions. Virus produced under EV-depleted conditions was more infectious. Changes in cellular metabolism and gene expression were associated with EV-depleted culture. The EV environment of HIV-1 infected cells appears to have a significant effect on virus production and infectivity. In cell lines of HIV-latency, significantly higher concentrations of p24 were observed in those cells cultured in EVD conditions. EV-dependence of cell cultures should be examined carefully prior to examining additional experimental variables. However, we also sound a cautionary note that direct actions of EVs may be accompanied by the effects of other, closely associated factors.

Dataset Information

CD9-HPLC-IAC Isolation of Serum Extracellular Vesicles for Proteomic Analysis

Publications

Column-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles.

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