Optimization of Extracellular Vesicles Isolation Protocol for Proteomics Analysis from Limited Cell Media Samples

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles secreted by cells that contain proteins, lipids, and nucleic acids reflective of their cell-of-origin. These vesicles, including exosomes, microvesicles, and apoptotic bodies, are pivotal in cell communication and have potential applications in liquid biopsies and therapeutic delivery. For proteomics analysis, isolating EVs from limited biological samples poses a challenge due to the limited amount of EV proteins and the complexity of media contaminants.

Here we sought to optimize an EV isolation workflow tailored for proteomics from small volumes (15 mL) of cell culture media. We compared two workflows: a combination of polymer-based precipitation followed by size exclusion chromatography (PPT+ExoSpin) and an ultrahigh-performance liquid chromatography-size exclusion chromatography (UHPLC-SEC) method.

Cell media from cell lines of pancreatic ductal adenocarcinoma (PDAC) were processed, and EVs were isolated using both workflows. The PPT+ExoSpin method eliminated the need for a concentration step of the EV fractions after SEC separation, significantly reducing EV loss. Isolated EVs were characterized via transmission electron microscopy (TEM) and western blot. The PPT+ExoSpin workflow demonstrated superior sensitivity, identifying a greater number of EV-associated proteins compared to the UHPLC-SEC method. Proteomics analysis revealed that the PPT+ExoSpin method identified 97 out of the top 100 proteins listed in the ExoCarta database, as well as all previously reported markers of PDAC EVs (EGFR, AXL, GPC1, EPCAM) in all replicates. An analogous analysis for EVs isolated using the UHPLC-SEC method identified only 68 or the 100 proteins in the ExoCarta database and either did not identify PDAC EV markers (EGFR and AXL) or did so with poor reproducibility (GPC1 – 2 replicates; EPCAM – 1 replicate).

The optimized PPT+ExoSpin workflow provides a reproducible and efficient approach for isolating EVs suitable for proteomics, enabling more comprehensive protein profiling from limited cell culture media volumes.