Abstract
Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.
Highlights
Exosomes are nanovesicles actively released by most epithelial cells to the extracellular milieu via the endo-exosomal pathway by exocytosis of multivesicular bodies (MVB) [1,2]
For the analysis, pooled urine samples were initially centrifuged at a relative centrifugal force (RCF) of 1,000 g for 20 minutes
Tamm-Horsfall Protein is the most abundant glycoprotein normally found in urine [29]
Summary
Exosomes are nanovesicles (diameter 40–100 nm) actively released by most epithelial cells to the extracellular milieu via the endo-exosomal pathway by exocytosis of multivesicular bodies (MVB) [1,2]. The discovery of exosome vesicles in urine [3] has rapidly opened new possibilities for the mechanistic understanding of biological processes and, importantly, has served as a source for novel biomarkers [4]. Exhaustive proteomic profiling of urinary exosomes has identified more than 1100 gene products, including 177 disease-related proteins derived from all nephron segments [5] and from the urogenital tract [6,7]. The identification from urine of distinct exosomal transcription factors [8] and nucleic acids encoding proteins native to all nephron segments [9] is groundbreaking, and highlights the need to precisely understand their biology which plausibly reflects new aspects of disease pathways. The treatment of the exosomal pellet obtained by the serial centrifugation protocol with dithiothreitol (DTT) has previously been proposed as a solution to reduce such interference [5]
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