2.1 Ethics Statement
This study was approved by the Bioethics Committee of Regional Specialized Hospital in Wrocław (approval ID: KB/nr 9/rok 2016). All samples were collected at Regional Specialized Hospital after obtaining written informed consent from study participants.
2.2 Description of Participants
Participants in this study included healthy volunteers (age range; 23 – 60, n=30, 16 men and 14 women), patients with biopsy-proven primary FSGS (age range 24-74, n=20, 16 men and 4 women), and primary IgAN (age range 21-66, n=19, 11 men and 8 women), patients with histopathologically confirmed primary ccRCC (age range 48-89, n=31, 18 men and 13 women), primary chRCC (age range 19-86, n=21, 12 men and 9 women), and prostate cancer (age range 57-79, n=7 men).
2.3 Urine Sample Collection and Handling
All participants provided first-morning urine (20 ml - 50 ml) in a sterile urine containers. Urine samples were obtained in the morning before scheduled renal biopsy (IgAN, FSGS patients) or surgery (renal cancer and prostate cancer patients). The specimens were processed at room temperature within 1-3 hours post-collection. All samples were tested for the following parameters: leukocyte esterase, nitrite, urobilinogen, protein, pH, blood, specific gravity, ketone, bilirubin, glucose with a Multistix 10 SG Reagent Strips (Siemens Healthcare Diagnostics Inc, NY, USA). Urine specimens of healthy controls and prostate cancer patients had normal urine parameters. The summary of urine dipstick test results is included in Supplementary table 1. Urine samples were centrifuged at 4300g for 30 minutes at room temperature and the supernatants were aliquoted into 2 ml sterile LoBind tubes, and stored at -80oC until further processing.
2.4 Sample Preparation for SWATH-MS/MS (quantitative analysis)
Urine supernatants were thawed in a waterbath (37oC) and vigorously vortexed. Equal volumes of urine supernatant from four subjects from the same group were pooled. The pooled samples were concentrated and desalted using 10-kDa molecular weight cutoff membranes (Merck KGaA, Darmstadt, Germany). Concentrated proteins were washed twice and eluted with equal volumes of molecular biology grade water (4000g/room temperature/20 minutes). The protein quantity in each sample was assessed in replicates by Qubit 2.0 fluorometer using Qubit Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Adequate volume of each pooled sample (corresponding to 15µg of proteins) was vacuum dried using speedvac (miVac, Genevac, UK). The protein pellets were suspended in 15 µl 50 mM NH4HCO3 and sonicated in a water bath (15 minutes/no heating). Samples were reduced by 10 mM dithiotreitol (DTT) at 56°C for 30 min, and subsequently alkylated by iodoacetamide in the final concentration of 20 mM (darkness/room temperature/30 minutes). After adding 20 µl of 50 mM NH4HCO3 to each sample, the samples were incubated overnight with MS-grade trypsin (1:50 trypsin to substrate ratio) with constant shaking (750 rpm) at 37oC. Digestion reaction was quenched by 5% formic acid/50% acetonitrile solution. The samples were desalted using a ZipTip C18 (Millipore) according to manufacturer`s instruction. Peptides were eluted into 50% acetonitrile/0,1% formic acid solution and subjected to LC-MS/MS.
2.5 Sample preparation for spectral library generation
The spectral library was generated by IDA-MS from the unfractionated peptide samples (prepared as in section ‘Sample Preparation for SWATH-MS/MS’) and fractionated peptide specimens.
Fractionated peptide samples were prepared from 120 µg and 100 µg protein pellets using Multiple Affinity Removal System (MARS-14, Agilent Technologies), and following the manufacturers` protocol. The low-abundant (LAP) and the high-abundant protein fraction (HAP) were subjected to proteolytic digestion and desalting as described in the section ‘Sample Preparation for SWATH-MS/MS’.
2.6 LC-MS/MS
The samples were separated with the Ekspert MicroLC 200 system (Eksigent, CA, USA) using ChromXP C18CL column (3 µm, 120 A, 150 x 0,5 mm). The injection volume was 5µl. The mobile phases consisted of LC-MS grade formic acid (0,1%) in water [A] and acetonitrile [B]. The separation was performed by a 30 minute gradient (0-2 min 10% B; 2-23 min 10-90% B; 23-28 min 90% B, 28,1-30 min 10% B). Data acquisition was performed with a TripleTOF 5600+ mass spectrometer with a DuoSpray Ion Source in positive ion mode (AB SCIEX, CA, USA). The microLC-MS/MS system was controlled by AB SCIEX Analyst TF 1.6 software.
- LC-MS/MS in IDA (information-dependent acquisition) mode
LC-MS/MS analyses in IDA mode were performed for the unfractionated and fractionated (LAP and HAP) samples. The TOF MS survey scan was conducted in the m/z range: 100-2000 with the accumulation time of 50 ms. Top 10 precursor ions, with the charge states from +2 to +5 were chosen for the collision-induced dissociation (CID) fragmentation. Product ion spectra were collected in the m/z range of 100-2000 with the accumulation time of 40 ms. The collision energy was automatically adjusted to the particular ion using rolling collision energy function. Precursor ions were excluded from reselection for 5 s after two occurrences. The duty cycle time was 1.11 seconds. The qualitative analyses in IDA mode were performed in three biological replicates, and three technical replicates per single biological replicate.
- LC-MS/MS in SWATH (Sequential Windowed of All Theoretical Fragment Ion Spectra) mode
The SWATH-MS analyses were performed for unfractionated protein samples. The parameters of SWATH-MS1 survey scan were as follows: high sensitivity mode, the m/z range: 100-2000, accumulation time of 50 ms. The parameters in the fragmentation mode were: m/z range 400-1000, divided into 25 windows, each 25 Da wide, accumulation time of 40 ms. The collision energy for each window was determined for a +2 to +5- ions centered upon the window with a spread of 2. The duty cycle time was 1.11 seconds. The quantitative SWATH analyses were performed in three biological replicates (each protein pool was subjected to three independent digestion and desalting procedures), and three technical replicates per each biological sample.
2.7 Data analysis
- Database search: IDA-MS
IDA MS/MS spectra were searched against SwissProt database (species: Homo sapiens, version 31.07.2017) by ProteinPilot (V4.5, SCIEX) using the Paragon algorithm. The search parameters were: TripleTOF 5600+ platform, Cys alkylation - iodoacetamide, digestion – trypsin, ID Focus: allow biological modifications, Search effort-thorough ID, Confidence>10%, automatic FDR (false discovery rate). Protein was considered a true positive if the following criteria were met: at least two peptides identified per protein with confidence >95% , and the FDR value < 0,1%.
- Data analysis: SWATH-MS
SWATH-MS data were processed against the MS/MS spectra libraries extracted from SwissProt database. The ProteinPilotgroup file was loaded into MS/MS All with SWATH Acquisition MicroApp 2.01 in PeakView 2.2 (SCIEX) to automatically generate a spectral library, according to the following parameters: maximum number of proteins-10000, modified peptides-allowed, peptides shared across proteins-not allowed, a maximum of 6 peptides per protein and 6 transitions per peptide. The MS/MS spectra and chromatograms of the chosen ions (XIC) from SWATH-MS analyses were compared with the spectral libraries. Starting parametres for the peptides were: [Conf] ≥ 99, FDR < 1%, XIC width: 75 ppm, offset XIC extraction window: 10 min. Retention time calibration was performed based on 3-7 peptides, uniformly distributed according to their elution time. The peptides and transitions were manually selected for further quantitative analyses.
- Statistical analysis
The t-test analysis were performed by MarkerView software version 1.2. (SCIEX).
2.8 ELISA
The expression level of RBP4 in urine supernatant of healthy subjects, FSGS, IgAN, renal cancer and prostate cancer patients was measured using Human RBP4 ELISA kit (Fine Test, Wuhan, China) following manufacturer`s instruction. The samples were diluted 3 times with Sample Dilution Buffer included in the kit. The urine creatinine levels were assessed in parallel with Creatinine (Cr) Colorimetric Assay Kit (Sarcosine oxidase method) (Elabscience Biotechnology, TX, USA) and QuantiChrom Creatinine Assay kit, DICT-500 (BioAssay Systems, CA, USA), both provided consistent results. The samples were diluted 30 and 10 times, respectively using double-distilled water. The level of uRBP4 (ng/ml) was normalized against the averaged amount of creatinine (ng/ml).
2.9 Statistical analysis of ELISA results
The box-whisker plots were done with Microsoft Excel 2013 (version 15.0.5207.1000, Microsoft Corp.). Statistical analysis was performed using the statistical package for the social scientists (SPSS version 16, IL, USA) (Pearson correlation test, nonparametric Mann-Whitney U Test with p < 0.05).