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Large-Scale Proteomic Analysis of Human Urinary Exosomes

Patricia A. Gonzales1, Trairak Pisitkun2, Robert Star3, Robert Kleta4, Nam Sun Wang1, and Mark A. Knepper2. (1) University of Maryland-College Park, 2113E Chemical & Nuclear Engineering Building, College Park, MD 20742, (2) Laboratory of Kidney and Electrolyte Metabolism; NHLBI; NIH, 10 CENTER DR; MSC 1603, Bethesda, MD 20892-1603, (3) Renal Diagnostics and Therapeutics Unit, NIDDK, NIH, Bethesda, MD 20892, (4) Human Biochemical Genetics, NHGRI, NIH, Bethesda, MD 20892

The goal of clinical proteomics is to develop cost-effective, non-invasive techniques for early disease detection, and for monitoring of therapy. In this study we have isolated exosomes from human urine and analyzed their proteome using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS, Thermo Electron Corporation, LTQ). Exosomes (<100 nm protein-containing vesicles secreted by epithelial cells lining the renal tubules and urinary drainage system) are isolated by differential centrifugation. The exosomes are solubilized in SDS and separated by SDS-PAGE. The gels are sliced into 40 separate pieces, each of which is treated with trypsin to proteolyze proteins into small peptide fragments. The peptides are introduced into the LC-MS/MS system and protein identifications are made from mass spectra using SEQUEST software to compare with theoretical spectra from GenBank sequence data. Previous studies of normal human urine had identified 304 proteins. In this new study of normal human urine, owing to the high sensitivity of the LTQ mass analyzer, we have 804 proteins despite stringent filtering of spectra, to avoid false positives. In addition, at least 36 proteins identified in this study, including many transporters, channels, ion pumps, and trafficking proteins, are already known to be associated with specific disease entities. We developed rabbit polyclonal antibodies to two of the identified proteins that are known to be mutated in Gitelman Syndrome (NCC) and Type 1 Bartter Syndrome (NKCC2). Examination of exosomes from patient urines with these syndromes revealed an absence of the relevant protein. Thus, we conclude that assays for mass spectrometrically-identified urinary protein biomarkers derived from exosomes have the potential for cost-effective disease identification.