Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

TLDR

Normal human urine contains abundant exosomes—40–100 nm vesicles derived from renal epithelial cells—and the resulting proteomic data are publicly available at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. The authors profiled urinary exosome proteins by LC‑MS/MS, extended the analysis to phosphoproteomics using neutral‑loss scanning to identify novel phosphorylation sites such as serine‑811 on NCC, and performed immunoblotting to confirm loss of NKCC2 in Bartter syndrome type I patient exosomes. The study identified 1,132 proteins in urinary exosomes, including 177 disease‑associated genes, uncovered numerous novel phosphorylation sites such as serine‑811 on NCC, and demonstrated that exosomes from Bartter syndrome type I patients lack NKCC2.

Abstract

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.