Two-Dimensional Size-Exclusion Chromatography-Based Isolation and Characterization of Plasma-Derived Extracellular...

Extracellular vesicles (EVs) collectively include nanosized vesicles and particles (condensates) that are released by cells and tissues and are circulated in blood and other physiological fluids. Their biomolecular cargo modulates key molecular and cellular pathways, rendering them a critical biological medium for advancing both diagnostic clinical applications and fundamental biology research. However, it is challenging to isolate highly heterogeneous EVs from complex biological matrices and fractionate EVs into subpopulations. A novel and straightforward two-dimensional size-exclusion chromatography (2D-SEC) approach for plasma-derived EV isolation and fractionation was developed as an alternative to existing low-purity EV isolation methods. The technique presented here enabled the isolation of EV subpopulation-enriched fractions while further depleting high-abundance plasma contaminants, resulting in improved detection of low-abundance EV-associated proteins. Furthermore, plasma-derived EV subpopulation-enriched fractions were isolated from prostate cancer (PCa) and healthy donors. The EV subpopulation-enriched fractions were subjected to mass spectrometry-based quantitative proteomic analysis to assess the presence of PCa- and tumor-associated proteins. Results show that deeper proteomic profiling of 2D-SEC EV isolates was achieved, compared to the 1D-SEC, identifying proteins involved in the PAM, MAPK, and NF-κB pathways, indicating that the developed 2D-SEC approach could be beneficial for enabling minimally invasive clinical applications and broader studies of EV biology.