Phosphoproteins in extracellular vesicles as candidate markers for breast cancer

Significance Protein phosphorylation is a major regulatory mechanism for many cellular functions, but no phosphoprotein in biofluids has been developed for disease diagnosis because of the presence of active phosphatases. This study presents a general strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs from small volumes of plasma samples and more than 100 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer as compared with healthy controls. This study demonstrates that the development of phosphoproteins in plasma EVs as disease biomarkers is highly feasible and may transform cancer screening and monitoring.