Impact of preanalytical factors on plasma extracellular vesicles and human plasma proteome

Using human plasma and its protein-rich extracellular vesicles (EVs) for proteomic biomarker discovery requires an in-depth understanding of the influences of pre-analytical procedures. This study systematically investigates the ramifications of high-speed centrifugation, freeze-thaw cycles, and prolonged storage on the plasma proteome and abundance of plasma EVs. Employing a Mag-Net-assisted workflow for high-throughput EV-based plasma proteomics, we find that high-speed centrifuging plasma samples at 8,000 g prior to analysis reduces both EV and protein numbers to only 30% of the original plasma, rendering numerous established EV markers undetectable. In contrast, multiple freeze-thaw cycles within a week have very minimal effect on proteome coverage, albeit with a reduced number of EVs and subtle shifts in the enrichment and depletion patterns of specific proteins. Long-term storage of plasma in freezer for years gradually and significantly diminishes proteome coverage with reduced EV counts and altered size distributions. These results caution the use of biobank samples for retrospective studies, particularly longitudinally collected blood specimens that have been preserved for years for EV-focused biomarker research, and underscore the imperative for stringent standardization of pre-analytical protocols to enhance the reliability and reproducibility of plasma EV proteomics.