Comparative Analysis of Extracellular Vesicle Isolation from Equine Serum and Plasma

ABSTRACT: To compare the efficiency, reproducibility, and proteomic profiles of extracellular vesicles (EVs) isolated from equine serum and plasma using size-exclusion chromatography (SEC) and differential ultracentrifugation (UC)+SEC, and to assess how the starting material influences EV cargo. Blood was collected from six healthy adult horses into EDTA tubes (plasma) and plain tubes (serum). Platelet-free plasma and serum were prepared by sequential centrifugation and filtration. EVs were isolated using SEC or UC followed by SEC. Vesicle morphology and size distribution were analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). EV proteins were identified by (LC-MS/MS) and subjected to functional enrichment analysis (g:Profiler) to compare unique protein sets between groups. SEC outperformed UC in reproducibility, protein yield, and ease of use. Serum + SEC yielded the most consistent proteomes, with the largest ≥3/6 replicate overlap (464 proteins) and reliable detection of canonical EV markers. TEM confirmed cup-shaped vesicles of 50–150 nm, and NTA showed a predominant particle population of 100–200 nm. Proteomic analysis revealed that serum-derived EVs were enriched in translation/proteostasis pathways (ribosomal proteins, chaperones, proteasome components), whereas plasma-derived EVs were enriched in immune, adhesion, cytoskeletal, and hemostasis pathways. Both the isolation methods and starting material strongly influence EV yield and cargo composition in horses. SEC, particularly applied to serum, provides a reproducible and high-quality EV proteome suitable for immune, adhesion, cytoskeletal, and hemostasis biomarkers discovery, while plasma remains relevant for studies targeting translation/proteostasis pathways. Standardizing matrix selection, isolation workflows, and pre-analytical conditions will improve reproducibility and translational potential in equine EV research.

INSTRUMENT(S):

ORGANISM(S): Equus Caballus (horse)

TISSUE(S): Blood Plasma, Blood Serum

SUBMITTER: Bianka Swiderska  

LAB HEAD: Olga Witkowska-Piłaszewicz

PROVIDER: PXD068077 | Pride | 2026-01-23

REPOSITORIES: Pride