A translational protocol for the analysis of plasma-derived extracellular vesicle proteomics

We establish here an EV purification and proteomic analysis workflow in a real world clinical setting in which we evaluate different variables, from blood collection through protein preparation for mass spectrometry (MS). We compare blood collection tubes, sample transportation and storage conditions, EV enrichment methods, size exclusion chromatography columns, as well as lysis and protein preparation conditions. For each parameter tested, we assess hemolysis, particle concentration and size, protein quantity, protein markers (EV-related, cell-specific, co-isolates) and, for some, comprehensive proteomic analysis using mass spectrometry.