Urine, by accumulating all kinds of changes, was proposed to be a better source for
biomarker discovery. As one of the most common post-translational modifications, phosphorylation plays a vital role in many
biological activities. However, the urine phosphoproteome has been largely neglected due to the low abundance of
phosphoproteins and the presence of various
phosphatases in urine. The low level of background phosphorylation in urine is actually advantageous, as urinary
phosphopeptides/
proteins that are stable to the
phosphatases present in urine have the potential to serve as valuable disease
biomarkers. Using a TiO2 enrichment strategy, this study aimed to create a comprehensive proteomic profile of human urinary
phosphoproteins and to characterize the changes in the urine phosphoproteome after incubation of urine with
renal carcinoma cell lysates. In total, 106 urine phosphorylation sites corresponding to 64
proteins, including 80 previously unidentified human urine
protein phosphorylation sites, were identified by mass spectrometry. Fifteen
phosphopeptides, together averaging 47% of the total
phosphopeptides, were found in samples from three individuals. Cellular
proteins are potential source of
biomarker in urine phosphorylated
proteins. Addition of
renal carcinoma cellular
proteins to urine did not significantly change the phosphorylation level of urine
proteins. But there were still a few
phosphopeptides from cell lysates survived urinary
phosphatases; such
phosphopeptides represent potential
biomarkers in urine.