Should urine pH be adjusted prior to gel-based proteome analysis?

The urine has become one of the most widely used clinical samples for biomarker discovery. The pH of human urine may vary largely from 4.5 to 8.0. Previously, it was questionable whether the urine pH would affect proteome analysis and whether the urine pH needed to be adjusted prior to proteome analysis remained unclear. We therefore performed a systematic analysis of the effect of urine pH on proteome profile. Midstream second morning and random afternoon urine samples were collected from 5 males and 5 females who were healthy and had no recent medication. After removal of cells and debris by low-speed centrifugation, pH levels of individual samples were measured and urinary proteins were isolated by 75% ethanol precipitation. Equally loaded 100 microg of proteins from individual samples were resolved in 2-DE (linear pH 3-10) and visualized with SYPRO Ruby fluorescence stain. There was no significant correlation between difference in the morning versus afternoon urine pH (DeltapH) and %match of protein spots derived from morning versus afternoon urine samples in individual samples (Pearson's r = 0.074; p = 0.839). In parallel, all individual samples with equal volume were pooled. The pH of the pooled urine was adjusted to 4-10 and urinary proteome profiles were analyzed as for individual samples. ANOVA with Tukey's posthoc multiple comparisons showed no significant differences in total number of detected spots and %match among various pH levels. Our data suggest that the urine pH has no significant effects on urinary proteome profile and thus needs no adjustment prior to gel-based proteome analysis.