Abstract: The state of protein phosphorylation and glycosylation can be key determinants of cellular physiology such as early stage cancer, but the development of phosphoproteins and/or glycoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate, for the first time, a strategy to isolate and identify phosphoproteins/glycoproteins 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 by isolating from small volume of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer than in healthy controls. Several novel biomarkers were validated in individual patients using Paralleled Reaction Monitoring for targeted quantitation. Similarly a group of glycoproteins in plasma EVs are identified.

Abstract: The state of protein phosphorylation and glycosylation can be key determinants of cellular physiology such as early stage cancer, but the development of phosphoproteins and/or glycoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate, for the first time, a strategy to isolate and identify phosphoproteins/glycoproteins in extracellular vehicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs by isolating from small volume of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer than in healthy controls. Several novel biomarkers were validated in individual patients using Paralleled Reaction Monitoring for targeted quantitation. Similarly a group of glycoproteins in plasma EVs are identified.

Abstract: Methods and chemicals for the capture and analysis of a selected group of protein modifications. Post-translational modifications alter the functional groups in proteins. The resulting modified proteome is useful for biomarker discovery, clinical diagnostics, and protein dynamics. Present immunoassays to quantify modified proteins are limited by their dependence on antibodies, which are often not specific for post-translational modifications. This invention utilizes a reagent and platform with a binding moiety that is capable of selectively binding to the modified protein.