Figure 3.

Evaluation of quantification performance in biological samples

(A) Pooled human plasma was serially diluted with exosome-depleted fetal bovine serum, and EVs were isolated from five replicates of each dilution level. Five separate SEC columns were used, one for each replicate dilution curve as indicated by the experimental schematic. Dilution curves fit with a linear regression are displayed for several select EV marker proteins. Average protein abundance values at each dilution point were used to fit the curve.

(B) Histogram representing mean R² values of the replicate dilution curves for all identified proteins showed that 850 proteins had an R² value >0.9.

(C) Analysis of the CVs of the five replicate measurements at each dilution point showed low variation at each dilution point.

(D) Linear regression showing the high correlation between summed EV protein intensity and dilution factor. Correlation is much higher when contaminants identified by using the co-migration-based filter process are removed prior to summing protein intensity (contaminants filtered, R² = 0.992 versus no filtering, R² = 0.815). High correlation indicates that total EV abundance can be estimated by summed EV protein intensity after contaminant filtering.