A New Approach to Monitoring Protein Transfer via Extracellular Vesicles

Extracellular vesicles (EVs) allow the exchange of protein, lipids and genetic material for communication between cells. We developed a method to track protein exchange between cells via EVs, using stable isotope labelling of amino acids in cell culture (SILAC). We compared EV isolation methods: ultracentrifugation and size exclusion chromatography (SEC) and undertook characterisation through nanoparticle tracking analysis (NTA), then optimised the requirement (0-10%) for foetal calf serum (FCS) and EV application concentration (6.5 million-200 EVs/cell seeded). We employed heavy amino acids L-Arg-HCl and L-Lys-2HCl to label donor cells and subsequent EV proteins. Unlabelled recipient cells were treated for 12 hours with EVs, at various concentrations. Mass spectrometry proteomics was used to assess uptake of heavy labelled EVs into recipient cells. A labelling efficiency of 62% was achieved. There was no relationship between the number of EVs applied to cells and the number of heavy proteins detected in the recipient cells. Pathway analysis indicated an inflammatory effect of applying EVs to cells, but the potential cause of this was unclear. We identified transferred protein cargo from EVs in recipient cells. Further optimisation of this method is still necessary.