Comparative Analysis of Extracellular Vesicle Isolation From Equine Serum and Plasma Using Two Isolation Methods With Structural and Proteomic Validation.

Extracellular vesicles (EVs) are promising biomarkers and mediators of intercellular communication, but their isolation from equine biofluids remains challenging. This study compared two isolation workflows-size-exclusion chromatography (SEC) and differential ultracentrifugation followed by SEC (UC + SEC)-to evaluate their efficiency, reproducibility, and the proteomic composition of EVs derived from equine serum and plasma. Blood from six healthy horses was processed to obtain platelet-free plasma and serum. EVs were isolated using SEC or UC + SEC and characterized by transmission electron microscopy, nanoparticle tracking analysis, and mass spectrometry-based proteomics with functional enrichment analysis. SEC provided higher reproducibility, greater protein yield, and a simpler workflow than UC + SEC. Serum combined with SEC yielded the most consistent proteomic profiles, with strong detection of typical EV markers and the largest overlap among replicates. Vesicles displayed the expected morphology and size distribution, with a predominant population between 100 and 200 nm. Plasma-derived EVs were enriched in proteins related to translation, chaperone activity, and proteasome function, while serum-derived EVs contained proteins involved in immune processes, cytoskeletal organization, adhesion, and hemostasis. Both the isolation method and biological matrix significantly influenced EV yield and proteome composition. SEC applied to serum provided a reproducible and high-quality EV preparation suitable for biomarker discovery. As this was a methodological comparison in healthy animals, diagnostic test performance metrics (sensitivity, specificity, PPV/NPV) were outside the scope of the study; our goal was to quantify upstream analytical determinants (matrix and isolation workflow) that influence reproducibility and discovery-phase proteomic readouts.