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

Area of Science:

• Veterinary Medicine
• Biochemistry
• Proteomics

Background:

• Extracellular vesicles (EVs) are crucial for intercellular communication and hold potential as biomarkers.
• Isolating high-quality EVs from equine biofluids presents significant challenges.
• Standardized methods are needed for reliable EV isolation and analysis in horses.

Purpose of the Study:

• To compare size-exclusion chromatography (SEC) and differential ultracentrifugation followed by SEC (UC+SEC) for EV isolation from equine serum and plasma.
• To evaluate the efficiency, reproducibility, and proteomic profiles of EVs obtained by different methods and matrices.
• To identify an optimal workflow for equine EV isolation for biomarker discovery.

Main Methods:

• Equine serum and platelet-free plasma were processed from six healthy horses.
• EVs were isolated using SEC and UC+SEC workflows.
• Characterization involved transmission electron microscopy, nanoparticle tracking analysis, and mass spectrometry-based proteomics.

Main Results:

• SEC demonstrated higher reproducibility, protein yield, and a simpler workflow compared to UC+SEC.
• Serum processed with SEC yielded the most consistent proteomic profiles with typical EV markers.
• EVs exhibited expected morphology and size (100-200 nm); proteomic content varied between serum and plasma sources.

Conclusions:

• The choice of isolation method and biological matrix significantly impacts EV yield and proteome composition.
• SEC applied to equine serum provides a reproducible, high-quality EV preparation suitable for biomarker discovery.
• This methodological comparison highlights key factors influencing EV analysis in horses.