Dataset Information

Proteomic Analysis of Bacterial Culture Supernatants

ABSTRACT: The supernatants from bacterial cultures were collected, concentrated by ultrafiltration (3 kDa cutoff), and subsequently reduced, alkylated, and digested with trypsin. Digested peptides were purified via SCX and StageTips, dried, and resuspended in 5% ACN with 0.1% TFA. LC-MS/MS analysis was performed using an Orbitrap QE Plus.

ORGANISM(S): Cellular Organisms

SUBMITTER: Takumi Nishiuchi

PROVIDER: PXD067284 | JPOST Repository | Wed Aug 13 00:00:00 BST 2025

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	BPcultured_20230220.raw	Raw
	peptides%20for%20Papain.xlsx	Xlsx

Items per page:

1 - 2 of 2

Similar Datasets

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.

Dataset Information

Proteomic Analysis of Bacterial Culture Supernatants

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets