Isolation of extracellular vesicles for proteomic profiling

Extracellular vesicles are nano-sized lipid bilayer vesicles released from most cells, including archaea, bacteria, and eukaryotic cells. These membrane vesicles play multiple roles in cell-to-cell communication, including immune modulation, angiogenesis, and transformation of cells by transferring genetic material and functional proteins. They contain specific subsets of proteins, DNA, RNA, and lipids that represent their cellular status. Furthermore, extracellular vesicles are enriched in cell type- or disease-specific vesicular proteins, especially plasma membrane proteins, which have pathophysiological functions; these vesicular proteins are considered novel diagnostic biomarkers as well as therapeutic targets. To profile the proteome, various purification methods of extracellular vesicles have been developed, but density gradient ultracentrifugation is considered the most promising. In this chapter, we describe the isolation of extracellular vesicles derived from SW480 cells and the preparation of tryptic peptides for mass-spectrometry-based proteomic analysis.