Tetraspanins are unevenly distributed across extracellular vesicles

Tetraspanins such as CD9, CD63, CD81 are standard for identifying extracellular vesicles. However, an important question to be asked is whether the amounts of these proteins is homogeneous across a distribution of EV's or across different populations. Some preliminary work [@mizenko2021Tetraspanins are unevenly distributed across single extracellular vesicles and bias sensitivity to multiplexed cancer biomarkers] shows that the number of tetraspanins can change. The image below are results using the ExoView instrument.

Essentially it is possible to see the cross-correlation between different proteins and their change over time (figure b). I don't really understand, for example, how can it be that if the capture antibody is CD9, it shows such a low signal when the detection antibody is also CD9. I wonder if this is a matter of efficiency, or that CD9 is a single-protein in most EV's (and thus can't be double bounded).

I wonder if it is possible to use syntenin-1 as a marker after the EVs have been immobilized on a substrate. For example by doing a two step measurement, first of the membrane proteins and then of the intraluminal proteins.

Moreover, the abundance of tetraspanins in EVs can be linked to the sample preparation protocol.