Sequential phosphoproteomics and N-glycoproteomics of plasma-derived extracellular vesicles

Extracellular vesicles (EVs) are increasingly recognized as important cargos for intercellular communication and promising sources for less invasive biomarker discovery. As the state of protein post-translational modifications (PTMs) such as phosphorylation and glycosylation can be a key determinant of cellular physiology such as early stage cancer, comprehensive characterization of protein PTMs in EVs are highly valuable, but significant challenges remain. Recently we demonstrated the feasibility to isolate and identify phosphoproteins and glycoproteins in EVs from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides and 1,500 unique glycopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phospho- and glyco-proteomics, we identified a number of phosphoproteins and glycoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer. Here we present an updated workflow to sequentially isolate phosphopeptides and N-glycopeptides, enabling multiple PTM analyses with the same clinical samples and presenting intriguing functional molecules for further validation.