Extracellular vesicles have become heavily researched in recent years for their roles in normal and diseased pathologies, including the areas of cancer, immunology and neuroscience. Although there has been rapid growth in EV research, a major hurdle in furthering our understanding of EVs is the lack of standardised approaches to EV isolation. In particular the impact of various EV isolation techniques in downstream analysis has not been well considered. This project focused on what protein differences might arise due to differences in isolation method.
The prostate bone metastasis cell line PC-3 was cultured for 48h in serum-free media before EV isolation. Media was collected and cleared of cellular debris before being passed through a 0.22µm syringe filter unit. Media was then passed through either a 0.1µm syringe, or 0.1µm vacuum filters to remove larger vesicles. EV isolation was then performed using either ultrafiltration or ultracentrifugation. Proteins were separated by electrophoresis, visualised with silver stain, and identified by LC/MSMS.
Differences were observed between isolation methods, indicating that the isolation techniques used for EVs can have an impact on downstream applications, particularly when considering proteomic analyses.