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Running title: Highly pure isolation of small extracellular vesicles from bovine plasma Title: Technical note: Efficient enrichment of plasma-derived extracellular vesicles from small volumes of bovine blood

ABSTRACT: This study presents a scalable method for isolating small extracellular vesicles (sEVs) according to MISEV (minimal information for studies of extracellular vesicles) guidelines from 4 mL of bovine plasma while achieving a high purity suitable for proteomics analysis. The protocol combines ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF) to minimize contamination by lipoproteins and free proteins. The purified sEVs were characterized through transmission electron microscopy (TEM), tunable resistive pulse sensing (TRPS), dynamic light scattering (DLS), and flow cytometry (FC), assessing particle size, morphology, and the presence of membrane markers such as CD9. The purity assessed by the particle-to-protein ratio (4.8 × 10⁸ particles/µg in average) was improved (× 10) compared to the previous protocol. Mass spectrometry (LC-MS/MS) and Parallel Reaction Monitoring (PRM) identified 417 proteins, 93% of which are referenced in sEVs databases. Specific markers of sEVs have been identified including cytosolic (HSPA8, SDCBP, ACT, TUB, and GAPDH) and membrane (CD9 and CD81) Markers. Gene Ontology (GO) analysis revealed an enrichment of sEVs proteins. This study demonstrates that this sEVs enrichment protocol is well-suited for small plasma volumes. These results pave the way for future studies of biomarker research on animal health and performance.

INSTRUMENT(S):

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Blood Plasma

SUBMITTER: Didier VIALA

LAB HEAD: BONNET Muriel

PROVIDER: PXD068315 | Pride | 2025-11-17

REPOSITORIES: Pride

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Efficient enrichment of plasma-derived extracellular vesicles from small volumes of bovine blood.

Prieur Vincent V Isabelle Cassar-Malek CM Arnaud Delavaud D Liam Barry-Carroll BC Jean-Christophe Delpech D Isabelle Morel M Sylvain Lerch L Didier Viala V Philip Chennell C Céline Boby B Muriel Bonnet B

Journal of animal science 20250101

There is a growing interest in small extracellular vesicles (sEVs). These nanoparticles, which range in diameter from 30 to 150 nm, are secreted by cells into their surrounding environment and transfer biological content to distant cells. However, the lack of consensus on sEV isolation, from bovine plasma limits their study. This work aimed to develop an optimized method to enrich sEVs from 4 mL of bovine blood plasma. To increase the yield of sEVs while reducing contamination from other particl ...[more]

PMID: 41206965

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