Urine from twenty-four healthy volunteers (38 ± 11 years old), including twelve males and twelve females.

For GELFrEE separation, urine samples were prepared using a protocol. Briefly, the pooled sample was fractionated in parallel using an eight-channel multiplexed GELFrEE 8100 Fractionation system (Protein Discovery, Knoxville, TN, USA). Application of 50 V for approximately 75 min and then 100 V for 105 min resulted in twelve GELFrEE fractions. The volume of each fraction was concentrated to approximately 125 μL using a SpeedVac Concentrator (Thermo Fisher Scientific, Asheville, NC, USA). Next, the samples underwent SDS removal using Pierce Detergent Removal Spin Columns (Pierce, Rockford, IL, USA). For LP-IEF fractionation, urinary proteins were desalted and cleaned using Amicon Ultrafiltration devices with a 10-kDa molecular weight cutoff (Merck Millipore Inc., Billerica, MA, USA). Then, the desalted urinary proteins were focused (approximately 2.5 h at 1 W) using a ten-chamber Microrotofor LP-IEF system (Bio-Rad, Hemel Hempstead, UK). Ten IEF fractions were collected; few protein bands appeared in fractions 7–10. Then fractions 6–10 were pooled into one sample.Urinary proteins were digested with trypsin (Trypsin Gold, mass spec grade, Promega, WI, USA) using filter-aided sample preparation methods. Proteins were loaded onto 10-kDa filter devices (Pall, Port Washington, NY, USA), and 8 M urea in 0.1 M Tris-HCl (pH 8.5) was added to wash the samples. The proteins were denatured by incubation with 50 mM dithiothreitol at 56 °C for 1 h and then alkylated in the dark for 45 min in 55 mM iodoacetamide. Trypsin was added (enzyme to protein ratio of 1:50), and the samples were incubated at 37 °C overnight. After digestion, the peptide mixtures were desalted on Oasis HLB cartridges (Waters, Milford, USA) and lyophilized for high-performance liquid chromatography separation.

Eighteen fractions that were separated by GELFrEE and LP-IEF and a pooled urine sample, were fractionated by offline high-pH RPLC columns (4.6 mm × 250 mm, C18, 3 μm; Waters Corp, Milford, USA). The samples were loaded onto the column in buffer A1 (10 mM NH4FA in H2O, pH = 10). The elution gradient was 5–30% buffer B1 (10 mM NH4FA in 90% acetonitrile, pH = 10; flow rate = 1 mL/min) for 60 min. The eluted peptides were collected at one fraction per minute. After lyophilization, the 60 fractions were re-suspended in 0.1% formic acid and concatenated into 20 fractions by combining fractions 1, 21, 41 and so on.

Each sample was analyzed on a reverse-phase C18 self-packed capillary LC column (75 μm × 100 mm, 3 μm). The elution gradient was 5–30% buffer B2 (0.1% formic acid, 99.9% acetonitrile; flow rate = 0.3 μL/min) for 100 min. A TripleTOF 5600 coupled with UPLC system was used to analyze the sample, and the MS data were acquired in a high-sensitivity mode using the following parameters: 30 data-dependent MS/MS scans per full scan; full scans were acquired at a resolution of 40,000 and MS/MS scans were acquired at 20,000; rolling collision energy; charge state screening (including precursors with +2 to +4 charge state);dynamic exclusion (exclusion duration 15 s); MS/MS scan range of 250–1800 m/z; and scan time of 50 ms.

The MS/MS data were processed using the Mascot software (version 2.3.02, Matrix Science, London, UK) and searched against the Swiss-Prot database (Homo sapiens, 20 267 sequences, 2013 07 version). The Mascot search results were filtered using the decoy database method in Scaffold (version 4.3.2, Proteome Software Inc., Portland, OR, USA).