Quick steps to get glioma extracellular vesicles surface membrane proteins mapping based on tryspin/Lysin C digestion followed by Amicon and shot gun proteomic: identification of ghost protein

Extracellular vesicles (EVs) are known to mediate intercellular communication and regulate a diverse range of crucial biological processes. Novel therapeutic strategies have emerged based on these EVs as biological nanoparticles. EVs is as a generic term for all secreted vesicles released including at least ectosomes, microvesicles, apoptotic bodies and exosomes. In order to determine if it is possible to get access to the protein present at the surface of the EVs, we developed first a quickstep strategy. We performed a Trypsin/Lys C digestion treatment for EVs pellet, followed by an Amicon filtration. This procedure sep-arates the EVs from peptides of protein aggregates and external part of EVs membrane proteins. After separation step, all the fractions have been subjected of proteomic analyses. Comparison between T-0h and T-6h, Trypsin/Lysin C digestion revealed an evolution of the surface EVs proteins. Some surface proteins have been demasked after 6h enzymatic digestion like CD81, CD82 ,Ust, Vcan, Lamp 1,rab43, Cspg4, Annexin A2, Synthenin 1,VSP37b. These results established an evolu-tion in time course of the nature of the surface proteins in glioma tumor.