Characterization of the human urinary proteome and peptidome

Urine represents an ideal source of clinically relevant biomarkers since it contains a large number of proteins and low molecular weight peptides. Characterization of the normal urinary proteome and peptidome can serve as a reference for disease and can aid in biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases. We developed an integrated proteomic and peptidomic analytical protocol for normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer. By analyzing six urines from healthy individuals, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma. Thus, this study can provide a reference for assessing alterations in cancer-specific biomarkers.