Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution-phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. Nearly, 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources.
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