Characterization of Protein Content Present in Exosomes Isolated from Conditioned Media and Urine

Cells secrete biomolecules into the extracellular space as a way of intercellular communication. Secreted proteins can act as ligands that engage specific receptors-on the same cell, nearby cells, or distant cells-and induce defined signaling pathways. Proteins and other biomolecules can also be packaged as cargo molecules within vesicles that are released to the extracellular space (termed extracellular vesicles or EVs). A subclass of such EVs, exosomes have been shown to horizontally transfer information. In recent years, exosomes have sparked tremendous interest in biological research, both for the discovery of novel biomarkers and for the identification of signaling molecules, as part of their cargo. Although multiple methods have been described for the isolation of exosomes, described here is a simple differential centrifugation approach that is well suited for the isolation of exosomes from conditioned cell culture media and urine. Mass spectrometry provides an ideal method to comprehensively analyze the protein cargo of exosomes. © 2017 by John Wiley & Sons, Inc.