Plasma has a substantial dynamic range. While depletion of high abundance proteins has been used often, this can remove important subsets of the proteome. We have developed a species-agnostic, antibody free approach to the isolation of circulating extracellular vesicles (EVs) from biofluids including serum and plasma.
The Mag-Net method does not rely on antibodies. Therefore, we are not capture specific subsets of extracellular vesicles. We see a size distribution that encompasses exosomes and microvesicles (~50 - 250 nm).
Additionally, we see enrichment of protein markers known in exosomes and microvesicles (B) and a siimultaneous depletion of abundant plasma proteins (B) that typically comprise the majority of measurable signal in total, undepleted plasma.
Every stage of the Mag-Net EV enrichment coupled to protein digestion including binding, washes, denaturation, reduction, alkylation, and digestion have been automated on the KingFisher (Both Flex and Apex models). This allows the protocol to be scaled to a 96-well plate format.
EVs are bound to ReSyn SAX beads at pH 6.3 and washed at pH 6.5 to reduce the binding of similarly sized but uncharged lipoproteins.
We have found that eluting EVs from the beads when slated for LC-MS analysis causes a significant loss in sample and sensitivity. In our workflow, the EVs remain bound to the beads and they are added directly to denaturing and reducing agents on the KingFisher system.
