Analyses of bloodborne nanoscale extracellular vesicles (nsEVs) have shown tremendous promise in enabling the development of noninvasive blood-based clinical diagnostic tests, predicting and monitoring the efficacy of treatment programs, and identifying new drug targets in the context of health conditions such as cancer and Alzheimer's disease. In this chapter we present a protocol for generating global nsEV proteomic profiles that can further the utility of nsEV analysis for the above biomedical applications by enlightening us of differences in protein abundance across normal and disease state nsEVs. This protocol features the use of magnetic particle-based immunoprecipitation to enrich highly purified populations of nsEVs directly from plasma or serum samples. The constituent proteins of these vesicles are subsequently characterized using a comparative shotgun proteomics approach that entails bottom-up, tandem mass spectrometric analysis of peptides generated by proteolytic digestion of nsEV-derived proteins. The methods described here are compatible with parallel processing of dozens of plasma or serum samples and can be valuable tools in enabling nsEV biomarker discoveries that have high translational relevance in the development of both novel therapeutics and blood sample diagnostic assays.
Keywords: Diagnostics; Drug discovery; Ectosome; Exosome; Extracellular vesicle; Immunoprecipitation; Liquid biopsy; Mass spectrometry; Proteomics.