Normal human urinary proteome variation by iTRAQ approach
Abstract
Urine is an important biofluids used for disease biomarker discovery. For differential proteomic analysis, it is essential to evaluate individual and gender variations. In this study, we characterized urinary proteomes of 14 healthy volunteers with regard to individual and gender variations using 2DLC-MS/MS analysis and 8-plex iTRAQ quantification. A total of 968 urinary proteins were identified, with 406 quantified in all individuals. The median inter-individual coefficients of variation (CVs) were 0.262 for urinary proteomes. Cluster analysis showed that male and female urinary proteomes exhibited different patterns. Further analysis revealed that individual variation was not correlated with protein abundance. The minimum sample size for proteomic analysis with a 2-fold change was 10 (4/5 for males/females using iTRAQ quantification) for urinary proteome.
In this study, we analyzed inter-individual and inter-gender variations in urinary proteomes by 2DLC/MS/MS and iTRAQ method. The second morning urine from 7 healthy male and 7 healthy female volunteers were used to evaluate variations in these urine proteomes. Each urinary sample was labeled with 8-plex iTRAQ regents and analyzed by 2DLC MS/MS; pooled urinary samples were used as controls. Technical variations, inter-individual variations, and inter-gender variations were analyzed. The minimum sample size needed for the urinary proteomes was evaluated for differential analyses.
| ID | age |
|---|---|
| m1 | 28 |
| m2 | 27 |
| m3 | 30 |
| m4 | 28 |
| m5 | 26 |
| m6 | 23 |
| m7 | 35 |
| f1 | 26 |
| f2 | 26 |
| f3 | 23 |
| f4 | 23 |
| f5 | 28 |
| f6 | 27 |
| f7 | 31 |
| Protocol | Description |
|---|---|
| Clinical materials |
Second morning urine samples were obtained from 7 healthy males and 7 healthy females (age 23–35 years, with a median age of 27 years). |
| Sample preparation |
The urinary samples were centrifuged at 5000 x g for 30 min, and the precipitates were removed. The supernatants were precipitated overnight at -4°C using 3 times the volume of ethanol. After 30 min of centrifugation at 10000 x g, the pellets were resuspended in lysis buffer (7 M urea, 2 M thiourea, 0.1 M DTT, and 50 mM Tris)；Each sample was digested using filter-aided sample preparation (FASP) method. Protein samples (200 ug) were reduced with 20 mM DTT at 37℃for 1 h and then carboxyamidomethylated with 50 mM IAA at room temperature in the dark for 45 min. Trypsin (4 ug) in 25 mM NH4HCO3 was added to protein samples for digestion overnight at 37°C. |
| iTRAQ Or TMT labeling |
The resulting peptides were mixed equally to create an internal standard and labeled with the 113 iTRAQ regent. The 7 male/female samples were individually labeled with 114, 115, 116, 117, 118, 119 and 121 iTRAQ regents according to the manufacturer’s protocol. |
| HPLC |
The pooled mixture of labeled samples was fractionated using a high-pH RPLC column from Waters (4.6 mm×250 mm, Xbridge C18, 3 μm). The samples were loaded onto the column in buffer A2 (H2O, pH = 10). The elution gradient was 5–30% buffer B2 (90% ACN, pH = 10; flow rate = 1 mL/min) for 60 min. The eluted peptides were collected at a rate of one fraction per minute, and the 60 dried fractions were re-suspended in 0.1% formic acid and pooled into 10 samples by combining fractions 1, 11, 21, 31, 41, 51; 2, 12, 22, 32, 42, 52; and so on. A total of40 fractions from the urinary and CSF peptide mixtures (male urine, female urine, male CSF and female CSF) were analyzed by LC-MS/MS. |
| LC/MS/MS |
Each fraction was analyzed using a reverse-phase C18 self-packed capillary LC column (75 μm×100 mm, 3 μm). The elution gradient was 5–30% buffer B1 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL/min) for 40 min. A Triple TOF 5600 mass spectrometer was used to analyze the eluted peptides from LC, and each fraction was run three times. The MS data were acquired under high-sensitivity mode using the following parameters: 30 data-dependent MS/ MS scans per full scan, full scans acquired at a resolution of 40,000 and MS/MS scans at a resolution of 20,000, rolling collision energy, charge-state screening (including precursors with a charge state of +2 to +4), dynamic exclusion (exclusion duration 15 s), an MS/MS scan range of 100–1800 m/z, and a scan time of100 ms. |
| Data processing |
Mascot (Matrix Science, London, UK; version 2.3.02) was used for database searching of the MS/MS results and was set up to search SwissProt human database (20227 entries)；For protein identification, Scaffold (version Scaffold_4.3.2, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. |