Large-scale proteomics and phosphoproteomics of urinary exosomes.
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/.
Gonzales
Patricia A
PA
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Pisitkun
Trairak
T
Hoffert
Jason D
JD
Tchapyjnikov
Dmitry
D
Star
Robert A
RA
Kleta
Robert
R
Wang
Nam Sun
NS
Knepper
Mark A
MA
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000328:Adult; D001477:Bartter Syndrome / Q000652:urine; D002853:Chromatography, Liquid / Q000379:methods; D055354:Exosomes / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D049971:Thiazides / Q000737:chemistry; D014556:Urine
D010750:Phosphoproteins; D020543:Proteome; D049971:Thiazides; D000251:Adenosine Triphosphatases