Urinary exosomes for protein biomarker research

Exosomes represent a distinct class of membrane nanovesicles of endocytic origin that are released to the extracellular microenvironment from diverse cell types under both physiological and pathological conditions. Remarkable roles of exosomes have been revealed in intercellular communication, immune regulation, infection, aging and cancer. Exosomes carry and transfer proteins, nucleic acids and lipids, and are ubiquitous in most biofluids, such as urine, plasma, cerebrospinal fluid, etc. Membrane vesicles secreted by the epithelial cells of the urinary tract hold the promise to be an excellent source of disease relevant cargo proteins. In clinical proteomics urine is one of the most attractive biofluids as it can be obtained non-invasively, in large quantities and is relatively stable. Current isolation methods however are not sufficiently proficient to produce urinary exosomes (UEs) at a purity grade and with reproducibility suitable for downstream LC-MS based quantitative proteomics applications. Consequently urinary exosome based protein biomarker research today exclusively relies on targeted protein studies (Table 1). This chapter describes the current state-of-the-art in exosome research in general and urinary exosomes in particular with a special focus on the potential of UEs in protein biomarker discovery. Recently we have developed an improved isolation/purification method based on double-cushion sucrose/D2O ultracentrifugation (Raj et al., 2011b). The method relies on the solubilization of the major impurities associated with UEs in a carefully selected buffer solution. The new method separates exosomes from the heavier membrane fragments and/or vesicles more efficiently than current protocols and is compatible with LC-MS-based quantitative proteomics workflow.