Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery.

Abstract

Background: Extracellular vesicles (EVs) are an important source of blood biomarkers and are emerging as next-generation therapeutics. Demonstrating the purity of isolated EVs is essential for applications ranging from proteomics-based biomarker discovery to biomanufacturing. In this study, we systematically evaluated multiple EV isolation methods for plasma and developed a scoring method to identify the approach best suited for proteomics.

Methods: Commonly used enrichment techniques, including size-exclusion chromatography (SEC) and precipitation-based methods, were compared against the starting plasma in terms of particle yield and size, proteomic overlap, depletion of abundant plasma proteins, and enrichment of EV markers and unique proteins. To enable rigorous purity assessment, we established a targeted parallel reaction monitoring (PRM) mass spectrometry assay that quantified key EV markers and contaminant proteins across preparations.

Results: Among the methods tested, SEC showed the greatest enrichment of EV markers and unique proteins, with the lowest level of contaminants, resulting in the highest overall purity scores. SEC also allowed for the detection of EV-free proteins. Other methods, by contrast, performed sub-optimally and were less reliable for proteomics-driven biomarker discovery.

Conclusions: SEC provides the most EV-enriched plasma isolates for proteomics information, with minimal contamination from plasma proteins. The PRM-based purity scoring offers an objective means of benchmarking EV preparations and may help standardize EV isolation quality for both biomarker discovery and therapeutic manufacturing.