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.

			Action	DRS
	CD9_EV1.pep.xml	Pepxml
	CD9_EV1.raw	Raw
	CD9_EV2.pep.xml	Pepxml
	CD9_EV2.raw	Raw
	CD9_EV3.pep.xml	Pepxml