From hundreds to thousands: Widening the normal human Urinome (1).

It is currently unknown how many proteins can be detected in urine. Improving the analytical approach would increase their number and potentially strengthen their predictive potential in diseases. We developed a combination of analytical procedures for maximizing sensitivity and reproducibility of normal human urinary proteome analysis based on ultracentrifugation, vesicles separation, combinatorial peptide ligand libraries (CPLL) and solvent removal of pigments. Proteins were identified by an Orbitrap Velos Mass Spectrometry. Overall, 3429 proteins were characterized: most components (1615) were contained in vesicles while the remaining 1794 were equally distributed among CPLL and butanol insoluble fractions. Several proteins were detected exclusively in one of the phases of the procedure, suggesting that each step is crucial in the fractionation strategy. Many (1724) proteins are described here whose presence in urine has never been reported and represents a potential source of information considering that urine is the unique site of excretion of products of interaction of metabolic processes. Improving the characterization of normal urinary proteome would also represent the basis for the analysis of urine biomarkers in human diseases. BIOLOGICAL SIGNIFICANCE: Sub-fractionating normal urine by successive steps (vesicle separation, CPLL and solvent treatments) allowed the identification of 3429 proteins, a relevant part (1724) being detected for the first time in urine. Several proteins of new description have been implicated in physiology pathways and in pathologies thus representing a potential source of new information on both metabolic processes and diseases.