Large-scale proteomics and phosphoproteomics of urinary exosomes

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/.

Figures

Figure 1.

Disease-related protein: NKCC2 and Bartter syndrome type I. (A) Details of clinical phenotype for patients with Bartter syndrome type I, patient 1 and patient 2. (B) Ultrasound images showing calcium deposits (white arrowheads) in the kidneys of patients 1 and 2. (C) Immunoblot of urinary exosomes samples from patient 1, patient 2, control 1, and control 2 using polyclonal rabbit anti-NKCC2 and NCC antibodies.

Figure 2.

Novel phosphorylation site in the NCC. The serine-811 on the NCC protein is phosphorylated. (A) The phosphorylation site on the peptide is denoted by an asterisk (*). (B) The neutral loss peak (NL) from the +2 mass spectrum and the site-determining ions b5, b6, y7, and y8.

Figure 3.

Detection of AQP2-S256 phosphorylation in urinary exosomes. IMCD, rat inner medullary collecting duct treated with dDAVP (V2R-selective vasopressin analog) for 30 min; exo (10 μg), is human urinary exosomes, 10 μg; exo (72 μg), human urinary exosomes, 72 μg.

Figure 4.

Differential centrifugation procedure for the isolation of urinary exosomes from urine.

Figure 5.

Spectrum generated by InsPecT for CHMP1A protein (NP_002759). The peptide sequence is RVYAENAIRK. The tag region for the b ions and the y ions are shown by the black solid lines.

Figure 6.

Criteria to disambiguate data set. (A) An unambiguous identification when a peptide sequence was a 100% match without gaps to one and only one protein. (B) An unambiguous identification when a peptide sequence was a 100% match without gaps to more than one protein, but these proteins are splice-variant products of one unique gene. (C) An ambiguous identification when a peptide sequence was a 100% match without gaps to more than one protein deriving from more than one gene, and the identification was based only on that single peptide.