Placental Samples
In this study, four normal human FT whole placentas and four TP were collected. All procedures were carried out in accordance with the ethical committee of Avicenna Research Institute (ARI) (ethical approval No: 1397.007) and with the revised version of Helsinki Declaration in 2013. Written informed consents were obtained from all subjects before clinical sampling. All the participants had a history of at least one previous successful healthy pregnancy and delivery and were selected from Caucasians living in Tehran / Iran after matching their ages (30 ± 2 and 33±2 years for first trimester and term placenta donors, respectively). The normal FT placentas were taken from totally healthy pregnant women who had referred to the hospital because of bleeding following induced abortion. TPs were obtained from women who needed to undergo elective caesarian section.
All pregnant women had normal body mass index and had no history of abortion; chronic or acute illness, used no medications before caesarian section or induced abortion. To minimize the potential impact of sex on proteome profile, all TPs belonged to male fetuses, however, the gender of aborted fetuses were unclear (gestational age< 12w). The mean gestational age of FT placentas were 10 ± 2 weeks and those of TPs were 38 ± 1 week. All placenta samples were analyzed by a pathologist and confirmed to be normal. The placentas were quickly put in cold phosphate-buffered saline (PBS) after collection, kept on ice and immediately transferred to the laboratory. From each placenta, four samples with 1 cm thickness from four directions (including both maternal and fetal sides) and one sample from the central part were cut using a sterile scalpel and pooled. The weight of each wet punch was about one gram. The samples were then washed several times in cold PBS to eliminate the contaminating blood. All samples were stored in liquid nitrogen until protein extraction.
Protein extraction and quantification
In each group (FT and TP), four frozen placenta samples were mixed and pulverized by cryogenic grinding with liquid nitrogen by using a chilled mortar and pestle. Pulverized sample (0.1gr) was homogenized in 1 mL lysis buffer containing 8 M urea, 2% w/v CHAPS, 2% dithiothreitol (DTT) in 5 mM Tris–HCl pH 7.6 and incubated on ice for 15min with gentle vortexing. Homogenate was centrifuged at 15000 g for 1 h at 4ºC. Supernatant was collected and its protein concentration was determined by 2-D Quant Kit (GE Healthcare, USA). Aliquoted samples were then stored at -20 Cº until being analyzed by 2D-PAGE.
Two-dimensional PAGE
Initially, the first dimension (iso electrofucing, IEF) of 2D-PAGE was carried out using 17cm linear immobilized pH gradient (IPG) strip with pH 3-10 (Bio-Rad) and processed. Based on the protein distribution pattern, the differences in proteome of FT and TP were mostly localized between pH range of 5 to 8. To this end, all subsequent experiments were done using 17 cm linear IPG strip with pH 5-8 (Bio-Rad). FT and TP protein extracts were run and stained simultaneously in a twin gel electrophoresis system (Bio-Rad) to minimize the variations. In-gel rehydration of IPG strips was done by using 200 and 500 μg protein extract for Colloidal Coomassie Stain (CCS) and silver nitrate staining, respectively. Protein sample was mixed with rehydration buffer (8 M urea, 2% CHAPS, 2% DTT, 2% IPG buffer and 0.001% bromophenol blue) to a final volume of 300 μl. After rehydration (~17 hr at room temperature), isoelectric focusing (IEF) was performed on the strips at 20ºC to reach a total of 50,000 Vh (PROTEAN IEF Cell, Bio-Rad). Subsequently, the strips were equilibrated in equilibration buffer (6 M urea, 50 mM Tris–HCl pH 8.8, 30% v/v glycerol, 2% SDS, and 0.001% bromophenol blue) containing 60 mM dithiothreitol (DTT) for 15 min at 37ºC followed by another 15 min incubation in equilibration buffer containing 135 mM iodoacetamide (IAA) at room temperature. Next, a twin gel electrophoresis system (PROTEAN II xi Cell, Bio-Rad) was used for the second dimension. The strips were placed on 8-15% gradient SDS-PAGE gels, sealed using 1% agarose, and run first at a constant voltage of 30 V for 15 min followed by 45 V constant voltage for the next ~ 12 h until the front line reached the bottom of the gels.
2D gels staining and spot selection
First and third-trimester placenta protein 2D gels were stained with CCS or silver nitrate. 2D image scanner and image master 2D platinum software, v 6 (Pharmacia, Uppsala, Sweden) were used for scanning gels and spot analysis, respectively. The same parameters were used for all gels to avoid variation in analyses. A single master gel image containing all spots was prepared in each group as the master gel. After determining the percentage of intensity (% intensity) for each spot, the mean intensities of the same spots on different gels were compared by student T-test using EXCEL software (Microsoft, version 2010) program and p values <0.05 were considered statistically significant. Among the 120 DEPs, 20 proteins with higher score including 18 proteins with dual expression patterns in both FTs and TPS and 2 proteins with unique expressions in FTs were selected for LC-MS/MS analysis. These spots were carefully punched out of CCS-stained gels followed by in-gel digested as described by Shevchenko et al. (15). Gel pieces were dehydrated in 50% Acetonitrile and rehydrated in 50 mM Tris pH8.0 + 10 mM DTT. Pieces were heated for 15 minutes at 65°C. Bands were then reduced by adding 15 mM iodoacetamide (IAA) and incubating 30 minutes in the dark at room temperature. Remaining IAA was quenched by the addition of 10 mM DTT. Pieces were dehydrated once more with 50% Acetonitrile and rehydrated in a Trypsin/LysC solution. Digestion was carried out overnight at 37°C. Peptides were purified by reversed phase extraction and analyzed by LC-MS.
LC-MS/MS Parameters
For LC-MS/MS, acquisition was performed with an ABSciex TripleTOF 5600 (ABSciex, Foster City, CA, USA) coupled to an electrospray interface with a 25 μm iD capillary and connected to an Eksigent μUHPLC (Eksigent, Redwood City, CA, USA). Analyst TF 1.7 software was adapted to control the apparatuses and data processing and acquisition. For the IDA mode, the source voltage was set to 5.5 kV and maintained at 325°C. Curtain gas pressure was set at 27 psi, gas one at 27 psi, and gas two at ten psi. The separation was done on a reversed-phase HALOC18-ES column 0.3 μm, i.d., 2.7 μm particles, 150mm long (Advance Materials Technology, Wilmington, DE) and maintained at 60°C. Samples were injected by loop overfilling into a 5μL loop. For the 60 minutes (16) LC gradient, the mobile phase consisted of solvent A (0.2% v/v formic acid and 3% DMSO v/v in water) and solvent B (0.2% v/v formic acid and 3% DMSO in EtOH) at a flow rate of 3 μl/ min (15, 16).
Bioinformatics Analysis
All data from LC-MS/MS runs were analyzed simultaneously with the Protein Pilot software to identify candidate proteins. To further investigate the biological significance of twenty differentially expressed proteins, we carried out GO and pathway enrichment analyses employing different online databases and software. For GO, Shiny GO v0.61 (http://bioinformatics.sdstate.edu/go) and PANTHER v15 (http://www.pantherdb.org) were used and pathway enrichment analysis was done using Reactome database (http://reactome.org). R software (version 3.6.1, https://www.r-project.org/) and packages GOplot and ggplot2 were applied for visualization of GO terms and enriched pathways, respectively.