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The state of protein modification can be a key determinant of cellular physiology such as early stage cancer.Here we demonstrate,for the first time,a strategy to isolate and identify glyco-and phospho-proteins in extracellular vesicles(EVs)from human plasma as potential markers to differentiate disease from healthy states.We identified thousands of peptides with post-translational modifications(PTMs)in EVs isolated from small volumes of plasma samples.Using label-free quantitative proteomics,we identified proteins with PTMs in plasma EVs that are significantly higher in patients diagnosed with breast cancer as compared to healthy controls.Several novel biomarkers were validated in individual patients using Paralleled Reaction Monitoring for targeted quantitation.I will discuss the feasibility in developing proteins of PTMs in plasma EV as disease biomarkers that may transform cancer screening and monitoring and its enormous challenges leading to the promising land.