A 96well ultrafiltration approach for the high-throughput proteome analysis of extracellular vesicles isolated from conditioned medium

1. Abstract Extracellular vesicles (EVs), nanoscale vesicles that are secreted by cells, are critical mediators of intercellular communication and play a crucial role in diverse pathologies such as cancer development. Therefore, EVs are regarded as having high potential in the clinic, both for diagnostic and therapeutic applications. Unfortunately, EVs reside in complex biofluids and their consistent isolation at sufficient purity for mass spectrometry-based proteomics has proven to be challenging, especially when increased high-throughput is required. Here, we describe the incorporation of our previously reported filter-aided EV enrichment (FAEVEr) strategy for the isolation of EVs from conditioned medium, from harvest to proteomic analysis completely to a streamlined 96well format. We compared our approach with ultracentrifugation, the most widely used method for EV enrichment, in terms of protein identifications, consistency, reproducibility and overall performance, including the invested time, resources and required expertise. In addition, our results show that including relative high percentages of TWEEN-20, a mild detergent, markedly improves the final purity of the EV proteome by removing the bulk of non-EV proteins (e.g. serum proteins) and significantly increases the number of identified transmembrane proteins. Moreover, our FAEVEr 96well strategy improves the overall reproducibility with a consistent number of protein identifications and decreased number of missing values across replicates. This promotes the validity and comparability between results, which is essential in both a clinical and research setting, where consistency is paramount. Competing Interest Statement The authors have declared no competing interest. 4. List of abbreviations - CM - Conditioned medium - dgUC - Density gradient ultracentrifugation - DMEM - Dulbecco’s Modified Eagle Medium - EDS - EV-depleted foetal bovine serum - EV - Extracellular vesicle - FAEVEr - Filter-aided extracellular vesicle enrichment - FBS - Foetal bovine serum - FT - Flow-through - HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LC-MS/MS - Liquid chromatography coupled tandem mass spectrometry - MISEV - Minimal information for studies of extracellular vesicles - MWCO - Molecular weight cut-off - NTA - Nanoparticle tracking analysis - PEG - Polyethylene glycol - PEI - Polyethyleneimine - PES - Polyethylene sulfone - rEV - Recombinant extracellular vesicles - RT - Room temperature - SDS - Sodium dodecyl sulphate - SEC - Size-exclusion chromatography - TEAB - Triethylammonium bicarbonate - TEM - Transmission electron microscopy - UC - Ultracentrifugation