Purification and Phosphoproteomic Analysis of Plasma-Derived Extracellular Vesicles

A successful phosphoproteomics analysis of extracellular vesicles (EVs) requires a unique approach, fine-tuned to address the challenges that have plagued plasma-based biomarker discovery. Here, I detail a procedure, which combines EVtrap-based high-recovery EV isolation, phase-transfer surfactant method for protein extraction, and PolyMAC-based enrichment of phosphopeptides. The combination of these methods provides a highly effective strategy for EV-based phosphoproteome analysis and leads to the discovery of novel phospho-markers previously undetectable.