FAEVEr 96well for high-throughput EV proteomics

ABSTRACT: Extracellular vesicles (EVs), nanoscale vesicles that are secreted by cells, are critical mediators for inter-cellular communication and play a crucial role in cancer development. As a result, EVs are regarded to have high potential value towards the clinic, both for diagnostic and therapeutic applications. Unfortunately, EVs reside in complex biofluids and their consistent isolation in adequate purities for bottom-up mass spectrometry has proven to be challenging, especially in high-throughput. Indeed, traditional isolation methods are often labor-intensive, not scalable and may vary in yield and purity. Here, we investigate the incorporation of the previously described filter-aided EV enrichment (FAEVEr) strategy in a streamlined 96well format for the isolation of EVs from conditioned medium, including the initial pre-clearing, the EV isolation and purification and even downstream sample preparation for proteomics. We evaluated our approach comparing the results with ultracentrifugation, still the most widely used method for EV enrichment, in terms of protein identifications, consistency, reproducibility and overall performance, including the invested time, resources and expertise. In addition, our results show that including relative high percentages of TWEEN-20, a mild detergent, positively affects the final purity of the EV proteome by removing the bulk of non-EV proteins (e.g. serum proteins) and significantly increasing the number of transmembrane protein identifications. Moreover, the FAEVEr 96well strategy improves the reproducibility with a consistent number of protein identifications and decreased number of missing values across the replicates. This reliability promotes the validity and comparability between experimental results, which is essential in both a clinical and research setting, where consistency is paramount.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

TISSUE(S): Primary Cell, Embryonic Stem Cell

SUBMITTER: Jarne Pauwels  

LAB HEAD: Kris Gevaert

PROVIDER: PXD059819 | Pride | 2025-07-10

REPOSITORIES: Pride