Dataset Information

Bovine uterine fluid extracellular vesicle proteome on different timepoints of the oestrous cycle

ABSTRACT: Healthy cow uterine fluid (UF) was collected from synchronized cattle at day 0, 7 and 16 after ovulation. Extracellular vesicles (EVs) were isolated from UF. UF-EVs from same cattle on different timepoints were subjected to LC-MS/MS. Additionally, one sample before EV purification was subjected to LC-MS/MS (corresponding EV sample is named 1D0).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Uterine Fluid

SUBMITTER: Johanna Piibor

LAB HEAD: Alireza Fazeli

PROVIDER: PXD040201 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle.

Piibor Johanna J Waldmann Andres A Dissanayake Keerthie K Andronowska Aneta A Ivask Marilin M Prasadani Madhusha M Kavak Ants A Kodithuwakku Suranga S Fazeli Alireza A

Molecular & cellular proteomics : MCP 20230909 11

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of i ...[more]

PMID: 37678639

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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.

Dataset Information

Bovine uterine fluid extracellular vesicle proteome on different timepoints of the oestrous cycle

Publications

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle.

Similar Datasets

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