A translational protocol optimizes the isolation of plasma-derived extracellular vesicle proteomics

In translational research and clinical routine, liquid biopsy is a promising tool to direct individually targeted treatments. Among the components of liquid biopsy, extracellular vesicles (EVs) carry manyfold molecular cargo and are increasingly being studied for biomarker identification. In order to identify potential confounding factors and determine optimal conditions when studying blood-derived EV proteins, the impact of pre-analytical variables needs to be assessed. Here we establish an EV enrichment for proteomic analysis workflow in a real-world clinical setting in which we evaluate variables from blood collection through protein preparation and storage for mass spectrometry (MS). We assess hemolysis, particle concentration and size, protein quantity, protein markers and comprehensive proteomic analysis using mass spectrometry to assess the influence of different pre-analytical variables like blood collection tubes, transportation of blood samples and delayed processing. Under these conditions, density gradient and size exclusion chromatography using Sepharose CL-4B show good EV enrichment. For MS, lysis with increased protease inhibitors shows high protein yields while TCA protein precipitation results in high numbers of identified proteins. In summary, we develop here an optimized protocol for the analysis of plasma EV-derived proteomics, evaluating pre-analytical variables relevant for implementation in a clinical setting.