Phosphoproteins in extracellular vehicles as candidate markers for breast cancer

The state of protein phosphorylation can be a key determinant of cellular physiology such as early stage cancer, but the development of phosphoproteins from biofluids for disease diagnosis remains elusive. Here we demonstrate, for the first time, a strategy to isolate and identify phosphoproteins in extracellular vehicles from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides, representing 2108 phosphoproteins, in EVs isolated from small volume of plasma samples. Using label-free quantitative proteomics, we quantified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer than in healthy controls. Multiple novel biomarkers were validated in 13 patients using Paralleled Reaction Monitoring and antibody- and non-antibody based assays. The study demonstrated that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.