Urine is correlated with glomerular filtration, tubular reabsorption and secretion. Because it accumulate changes from both plasma and related organs, urine is considered to be one of the most attractive sources for biomarker studies. Urinary proteomic studies have discovered many candidate biomarkers for various diseases. In the biomarker discovery process, it is essential to comprehensively profile a normal urinary proteome as a baseline reference.
To achieve maximal urinary proteome coverage and to provide detailed information about the effects of different separation methods, one-, two- and three-dimensional separation strategies were used in this study. For one-dimensional (1D) separation, the digested urinary peptides were directly analyzed by 1D liquid chromatography-tandem mass spectrometry (LC/MS/MS); for two-dimensional (2D) separation, urinary peptides were fractionated by offline high-pH reverse-phase liquid chromatography (RPLC) and then analyzed by 1DLC/MS/MS; for three-dimensional (3D) separation, urinary proteins were first fractionated by gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) or liquid-phase isoelectric focusing (LP-IEF). Next, urinary peptides digested from each GELFrEE/LP-IEF fraction were further fractionated by RPLC just as with 2D separation and finally analyzed by 1DLC/MS/MS. In total, the whole urine proteome dataset eventually contained 3,048,648 spectra, 374,567 unique peptides and 6,412 proteins with protein FDR<1% and at least two unique peptides from all 383 runs.