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

Extracellular vesicles (EVs) are increasingly being recognized as important vehicles for intercellular communication and as promising sources for biomarker discovery. Because the state of protein post-translational modifications (PTMs) such as phosphorylation and glycosylation can be a key determinant of cellular physiology, comprehensive characterization of protein PTMs in EVs can be particularly valuable for early-stage diagnostics and monitoring of disease status. However, the analysis of PTMs in EVs has been complicated by limited amounts of purified EVs, low-abundance PTM proteins, and interference from proteins and metabolites in biofluids. Recently, we developed an approach to isolate phosphoproteins and glycoproteins in EVs from small volumes of human plasma that enabled us to identify nearly 10,000 unique phosphopeptides and 1,500 unique N-glycopeptides. The approach demonstrated the feasibility of using these data to identify potential markers to differentiate disease from healthy states. Here we present an updated workflow to sequentially isolate phosphopeptides and N-glycopeptides, enabling multiple PTM analyses of the same clinical samples. In this updated workflow, we have improved the reproducibility and efficiency of EV isolation, protein extraction, and phosphopeptide/N-glycopeptide enrichment to achieve sensitive analyses of low-abundance PTMs in EVs isolated from 1 mL of plasma. The modularity of the workflow also allows for the characterization of phospho- or glycopeptides only and enables additional analysis of total proteomes and other PTMs of interest. After blood collection, the protocol takes 2 d, including EV isolation, PTM/peptide enrichment, mass spectrometry analysis, and data quantification. This protocol describes a mass spectrometry-based workflow for combined analysis of protein phosporylation and N-glycosylation of extracellular vesicles obtained from a single blood plasma sample.