The endometrial tissue was obtained from 3 patients of PCOS and 3 healthy volunteers who already have children. Patients with PCOS took letrozole on the third day of menstruation, then ovulation has been continuously monitored since the 10th day of menstruation, and the endometrium was obtained on the fifth day after ovulation.
They were also screened for glucose metabolism and endocrine normality with serum determinations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), Estradiol, glucose and insulin on day 3 of the menstrual cycle. No participants showed any evidence of chromosomal abnormality, pathological uterine disorder or endometrial hyperplasia. None had used oral contraception or hormonal therapy during the previous 3 months. The diagnosis of PCOS was according to the 2003 Rotterdam criteria which included any two or all three of the following features:① oligo-/anovulation; ②clinical or biochemical signs of hyperandrogenism; ③polycystic ovary morphology on ultrasound[5]. The main demographic characteristics of the patient and control groups are summarized in Table 1; the PCOS and control groups did not differ regarding age, body mass index (BMI), FSH, LH and testosterone but did differ regarding insulin and glucose. Each biopsy was dry frozen at − 80°C for protein extraction. The patients were recruited at the Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, approved by the institutional ethics committee (No: 20170609). All patients gave informed consent prior to entering the study. Figure 1 shows the basic principle of iTRAQ quantitative proteomics and the main steps of quantitative techniques.
1.1 Protein Extraction
We used lysis buffer 3 (8 M Urea, TEAB or 40 mM Tris-HCl with 1mM PMSF, 2mM EDTA and 10mM DTT, pH 8.5) and two magnetic beads to extract proteins. Then we removed the mixtures into a tissue lyser for 2 minutes at 50Hz to release proteins. After that, the supernatant was transferred into a new tube after centrifugation with 25000g at 4℃ for 20 minutes, and reduced with 10 mM dithiothreitol (DTT) at 56 ºC for 1 hour and alkylated by 55 mM iodoacetamide (IAM) in the dark at room temperature for 45 minutes. Following centrifugation, the supernatant containing proteins was quantified by Bradford.
1.2 QC of Protein Extraction
(1) Protein Quantitation with Bradford assay
First of all, we added 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18µl BSA solution separately into a 96-well plate, then separately added 20, 18, 16, 14, 12, 10, 8, 6, 4 and 2µl pure water into the corresponding wells. At the same time, we also maked serial dilutions (20µl each well) of the unknown sample to be measured. After that, we added 180µl of coomassie blue into each well and mixed. Read the absorbance of each standard and sample well at 595 nm. Each sample had at least two duplicates. Then the absorbance of the standards vs. their concentration would be plotted. The we calculated the extinction coefficient and the concentrations of the unknown samples.
(2) SDS-PAG
15–30µg proteins were mixed with loading buffer in the centrifuge tube and heated at 95℃ for 5 minutes. Then, the mixture would be centrifuged at 25000g for 5 minutes, then the supernatant would be loaded into sample holes in 12% polyacrylamide gel. Run SDS-PAGE in constant voltage at 120V for 120 minutes. Once it finished, stain gel with coomassie blue for 2 hours, then add destaining solution (40% ethanol and 10% acetic acid) and put it on a shaker (exchange destaining solution for 3 ~ 5 times, 30 minutes a time).
1.3 Protein Digestion
The protein solution (100ug) with 8M urea was diluted 4 times with 100mM TEAB. We applied the trypsin gold (Promega, Madison, WI, USA) to digest the proteins (protein: trypsin = 40:1) at 37°C overnight. After that, we used a Strata X C18 column (Phenomenex) and vacuum-dried to desalt peptides according to the manufacturer's protocol.
1.4 Peptide Labeling
We dissolved the peptides in 30 ul 0.5 M TEAB with vortexing. After the iTRAQ labeling reagents were recovered to ambient temperature, they were transferred and combined with proper samples. We performed the peptide labeling by iTRAQ Reagent 8-plex Kit on the basis of the manufacturer's protocol. Then we combined and desalted the labeled peptides with a Strata X C18 column and vacuum-dried based on the manufacturer's protocol.
1.5 Peptide Fractionation
We separated the peptides through a Shimadzu LC-20AB HPLC Pump system coupled with a high pH RP column. After that, we reconstituted the peptides with buffer A (5% ACN,95% H2O, adjust pH to 9.8 with ammonia) to 2 ml and loaded it onto a column containing 5-µm particles (Phenomenex). Then we separated the peptides at a flow rate of 1 mL/min with a gradient of 5% buffer B (5% H2O, 95% ACN, adjust pH to 9.8 with ammonia) for 10 minutes, 5–35% buffer B for 40 minutes, and 35–95% buffer B for 1 minute. Then the system was maintained in 95% buffer B for another 3 minutes and decreased to 5% within 1 minute before equilibrating with 5% buffer B for 10 minutes. Next, we monitored the elution through measuring absorbance at 214 nm, and collected the fractions every one minute. Finally, we divided the eluted peptides into 20 fractions and vacuum-dried them.
1.6 HPLC
First of all, each fraction was resuspended in buffer A (2% ACN, 0.1% FA) and centrifuged at 20,000g for 10 minutes. Then the supernatant would be loaded on Thermo Scientific™ UltiMate™ 3000 UHPLC system which equipped with a trap and an analytical column. We loaded the samples on a trap column at 5µL/min for 8 minutes, and then eluted it into the homemade nanocapillary C18 column (ID 75µm x 25 cm, 3µm particles) with a 300nl/min flow rate. The gradient of buffer B (98% ACN, 0.1% FA) was raised from 5–25% in 40 minutes, and then raised to 35% in 5 minutes, followed by 2 minutes linear gradient to 80%, then maintained at 80% B for another 2 minutes, and finally returned to 5% in 1 minute and equilibrated for 6 minutes.
1.7 Mass Spectrometer Detection
We subjected the peptides separated from nanoHPLC into the tandem mass spectrometry Q EXACTIVE HF X (Thermo Fisher Scientific, San Jose, CA) for DDA (data-dependent acquisition) detection by nano-electrospray ionization. The relevant parameters of the MS analysis are presented as following: precursor scan range: 350–1500 m/z at a resolution of 60000 in Orbitrap; electrospray voltage: 2.0 kV; MS/MS fragment scan range: >100 m/z at a resolution of 15000 in HCD mode; normalized collision energy setting: 30 %; dynamic Exclusion time: 30 s; automatic gain control (AGC) for full MS target and MS2 target: 3e6 and1e5, respectively; The number of MS/MS scans following one MS scan: 20 most abundant precursor ions above a threshold ion count of 10000.
2. Bioinformatics Pipeline
After getting raw data, we will do each bioinformitics analysis as the client appoints on contract. Figure 2 demonstrates a complete pipeline for iTRAQ (Quantification) project.
3. Protein Quantification
We applied an automated software called IQuant for quantitatively analyzing the labeled peptides with isobaric tags[6]. It integrates Mascot Percolator[7] to provide reliable significance measures. In order to assess the confidence of peptides, the PSMs were pre-filtered at a PSM-level FDR of 1%. Then based on the "simple principle" (The parsimony principle), identified peptide sequences were assembled into a set of confident proteins. In order to control the rate of false-positive at protein level, a protein FDR at 1%, which is based on picked protein FDR strategy[8], would also be estimated after protein inference (Protein-level FDR < = 0.01). The process of the protein quantification comprised the following steps: Protein identification, Tag impurity correction, Data normalization, Missing value imputation, Protein ratio calculation, Statistical analysis, Results presentation.