Abstract
The protein content of urinary extracellular vesicles (EVs) is considered to be an attractive non-invasive biomarker source. However, little is known about the consistency and variability of urinary EV proteins within and between individuals over a longer time-period. Here, we evaluated the stability of the urinary EV proteomes of 8 healthy individuals at 9 timepoints over 6 months using data-independent-acquisition mass spectrometry. The 1802 identified proteins had a high correlation amongst all samples, with 40% of the proteome detected in every sample and 90% detected in more than 1 individual at all timepoints. Unsupervised analysis of top 10% most variable proteins yielded person-specific profiles. The core EV-protein-interaction network of 516 proteins detected in all measured samples revealed sub-clusters involved in the biological processes of G-protein signaling, cytoskeletal transport, cellular energy metabolism and immunity. Furthermore, gender-specific expression patterns were detected in the urinary EV proteome. Our findings indicate that the urinary EV proteome is stable in longitudinal samples of healthy subjects over a prolonged time-period, further underscoring its potential for reliable non-invasive diagnostic/prognostic biomarkers.
Highlights
The protein content of urinary extracellular vesicles (EVs) is considered to be an attractive noninvasive biomarker source
We assessed the stability of the urinary EV proteome using a highly reproducible generation proteomics approach based on liquid chromatography on-line coupled to data-independent acquisition (DIA)-MS15–17
The level of CD9, CD63 and CD81 exosome markers was more variable compared to the heat-shock proteins (HSPs), most notably between different individuals (Fig. 1d), with donor “Male5” showing the highest expression, suggesting that inter-individual differences in EV subpopulations might be present in the urine
Summary
The protein content of urinary extracellular vesicles (EVs) is considered to be an attractive noninvasive biomarker source. We assessed the stability of the urinary EV proteome using a highly reproducible generation proteomics approach based on liquid chromatography on-line coupled to data-independent acquisition (DIA)-MS15–17 This mass spectrometry approach combines the benefits of global discovery proteomics with the quantitative precision of targeted mass spectrometry with dynamic range over 3 order of m agnitude[17,18], and provides a pathway for large-scale clinical p roteomics[19]. To this end, we measured a longitudinal cohort of eight different healthy subjects (four females, four males) of whom urine samples were collected at 9 different timepoints over 6 months (see Fig. 1a for a schematic overview). The missing values (29,913 out of total 120,734 data points) are gray. (d) Expression levels of selected EV-related protein markers for each sample with Heat-shock proteins (upper panel), tetraspanins (middle panel), and TSG101 and PDCD6IP (lower panel) showing a good consistency in the level of these proteins in time
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