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Extracellular vesicles (EV) efficiently convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected twelve EV related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV producing cells and both parent cells as well as the recovered vesicles were characterised biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP tagged molecules per vesicle. The co-expression of each target protein with CD63 was further quantified by confocal imaging of single vesicles after double transfection of parent cells. We observed highly different loading efficiencies and specificities for the different proteins into EVs. Even for the most abundant Tetraspanins, the molecular levels in the vesicles did not exceed ca 30-40 fluorescent proteins per vesicle despite overexpression in the cells. Some of the GFP tagged EV reporters were either non-vesicular, despite co-purification with EVs, showed quenched fluorescence or comprised a significant …