2.1 Animal Model
Animal handling protocols were sanctioned by the Zhejiang University Committee on Animal Care and Use, while experiments adhered to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Adult male Sprague-Dawley rats (6 to 8 wk old) were procured from Experimental Animals Center of Zhejiang University (Hangzhou, China) and housed in our laboratory under standard conditions and freely provided with food and water. A preeclampsia model was developed as before [13–16]. Briefly, 10 pregnant rats were divided into two groups as follows: one group were treated with L-NAME (50 mg/kg) while the controls received saline via osmotic minipumps starting at day 17 of gestation until day 7postpartum. A total of 22 and 24 controls and L-NAME-exposed pups were born, respectively, and weaned at 21 days. Thoracic aortic of rats from two different groups were dissected, and blood was removed by rinsing. Thoracic aortic specimen were frozen at − 80°C.
Determination of blood pressure in 1-year-old rats was done with the tail-cuff method. Approximately 2-mm segments of mesenteric arteries (in the second-order branch of the superior mesenteric artery) were cut to remove fats and connective tissues, followed by mounting on a wire myograph chamber (DMT 620M; Aarhus N, Denmark), according to the manufacturer's recommendations. Briefly, arterial vessel segments were soaked in physiological Krebs' buffer that had been passed through 95% oxygen/ 5% carbon dioxide (pH 7.4), and incubated at 37℃. The contents were maintained in an equilibration period for 30 min, vessel tension elevated to 1 mN, followed by establishment of resting tension for 30 min. The KPSS was used to cause maximum contraction of the mesenteric arteries, which were then subjected to varying concentrations of vasoconstrictor phenylephrine (Phe) (10 − 9 to 10 − 5 mol/L). The resulting contraction was presented as % of contraction by 120 mm. Thereafter, a stable contraction plateau was established by Phe (1 µm)-treatment of mesenteric arterial rings to determine acetylcholine-induced vasodilatation. Subsequently, they were treated with varying concentrations of an actylcholine (10 − 8 to 10 − 5 mol/L). The readings are displayed as a % of precontraction relative to Phe.
2.2 Sample Preparation
Approximately 100 mg of thoracic aortic specimen were first crushed to form a fine powder, under liquid nitrogen, then mixed with a buffer comprising 4% SDS, 1 mM DTT, and 150 mM Tris-HCl (pH 8.0), to extract total proteins. The BCA protein assay (Pierce, Rockford, IL, USA) was carried out to quantify the amount of proteins extracted.
2.3 Protein Digestion and iTRAQ Labeling
Proteins were digested using a previously described protocol [17, 18]. About 200 µg of total protein samples were suspended into 30 µL of the aforementioned extraction buffer, supplemented with 100 mM dithiotreitol solution, and subjected 95℃ heating for 5 minutes. Sample were cooled subjected to ultrafiltration process (cutoff 10 kDa, Sartorius, Goettingen, Germany) with 200 µL UT buffer(150 mM Tris-HCl, and 8 M Urea, pH 8.0). They were then subjected to centrifugation for 30 min at 14,000 g at 20℃. Next, reduced cysteines were blocked with 100 µL of 50 mM iodoacetamide dissolved in UT buffer), and then subjected to a 20-min incubation in darkness. The resultant filtrate was centrifuged at 14,000 g for 20 min at 20℃, and the washed two times with 100 µL UT buffer at 14,000g for 20 min. Subsequently, the filtrate was mixed with 100 µL dissolution buffer (AB Sciex, Framingham, MA, USA), centrifuged at the aforementioned speed and temperature for 30 min. This procedure was carried out twice. The obtained filtrate was incubated overnight with 2 µg trypsin in 40 µL of trypsin at 37℃. The filtered units were separately put into fresh tubes and spun at 14,000 g at 20℃for 30 min, then peptide concentrations determined using UV light spectral density at OD280 .
We labeled the resultant peptide mixture with 8-plex iTRAQ chemical (AB Sciex, Framingham, MA, USA). Four thoracic aortic specimens belonging to control group (C) were labeled with a mass of 113, 114, 115 and 116 isobaric iTRAQ tags, while four corresponding preeclamptic tissues were labeled with mass 117, 118, 119 and 121 isobaric iTRAQ tags. The specimens, together with labeling solution, were for 2 h at room temperature prior to subsequent characterization.
2.4 Strong Cationic-exchange Chromatographic Separation
This procedure was carried out according to a previous protocol . Briefly, we acidified the combined sample using 1% trifluoroacetic acid, then using a PolySULFOETHYL column (4.6 × 100 mm, 5 µm, 200 Å, Poly LC Inc., Columbia, MD, USA), it was exposed to strong cationic-exchange chromatography (SCX) fractionation. Solvent A contained 10 mM KH2PO4 in 25% (v/v) ACN, whereas solvent B contained solvent A enriched with 500 mM KCl. Application of solvent A and B was done at the following gradients: 0–10% solvent B for 2 min, 10–20% solvent B for 25 min, 20–45% solvent B for 5 min, and 50–100% solvent B for 5 min. The collected fractions (at each minute) were analyzed by measuring the absorbance at 214 nm. The final step involved combining all samples to form 10 fractions, according to the quantity of peptides, followed by desalting using C18 cartridges (Sigma). Each SCX salt step fraction was dried under vacuum centrifugation, after which it was suspended in 40 µL 0.1% (v/v) trifluoroacetic acid.
2.5 LC - ESI- MS/MS analysis
LC-ESI- MS/MS Analysis, described previously , was carried out with 5 µg of peptide mixtures from each fraction analyzed using nano LC-MS/MS. Summarily, peptide mixtures were loaded into a Thermo EASY-nLC column, measuring 100 mm× 75 µm,3 µm, (Thermo Finnigan, San Jose, CA, USA) in solvent C (0.1% Formic acid), and separated using a linear gradient comprising solvent D (80% acetonitrile with 0.1% (v/v) formic acid) at a flow rate of 300 nL/min over 120 min: 0-100 min with 0–45% solvent D; 100–108 min with 45–100% solvent D; 108–120 min with 100% solvent D.
Data in the positive ion mode was acquired via the Q-Exactive (Thermo Finnigan, San Jose, CA, USA) mass spectrometer, at a selected mass range of 300–800 mass/charge (m/Z). Moreover, dynamic exclusion was used with 40.0 s duration, while Q-Exactive survey scans were set at resolutions of 70,000 and 17,500 at m/z 200 for HCD spectra. MS/MS data was obtained using the data-dependent acquisition method, targeting the top 10 most abundant precursor ions. Normalized collision energy was set at 30 eV whereas the underfill ratio on the Q-Exactive was defined as 0.1%.
2.6 Protein identification and quantification
Proteins were identified and quantified using a previously reported protocol . Briefly, proteins were first detected using the MASCOT search engine (version 2.2.1; Matrix Science, London, UK), embedded in the Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA, USA). This was achieved by searching the Uniport database of rat protein sequences (08-2013, downloaded from: http://www.uniprot.org/) as well as a decoy database, using the following search parameters: monoisotopic mass, peptide mass tolerance of ± 20 ppm; fragment mass tolerance of 0.1 Da; trypsin as the target enzyme; and allowing up to two missed cleavages. iTRAQ 8-plex labeled tyrosine and methionine oxidation were considered as variable modifications, whereas fixed modifications were considered as carbamidomethylation on cysteine, N-term of peptides labeled by iTRAQ 8-plex, and lysine. We set the False discovery rate (FDR) for both protein and peptide identification at less than 0.01, and this detection was supported by at least one unique peptide identification.
2.7 Bioinformatics analyses
We carefully analyzed significant differentially expressed proteins (p < 0.05), then selected and retained those with differential expression ratios above ± 1.2. We then used hierarchical cluster analysis, based on Cluster 3.0 and Java Treeview softwares, to determine the value of the resulting DEPs in differentiating our two experimental.
Thereafter, we performed disease and pathway analyses, as well as network generation, using the Ingenuity Pathway Analysis (IPA) software (QIAGEN, Redwood 185 City, CA), a Generally, IPA database that relies on available publications describing biological mechanisms, interaction and functions of protein. Here, calculating z-scores can be used to infer activation states (“inhibited” or “activated”) of related biological processes. The Fisher’s exact test was employed to determine p-values, hence predict the likelihood of association among proteins in some datasets. The resulting biological process was only attributed to chance.
2.8 Western blot assay
Determination of protein expression was performed using Western blot assay as previously described . Briefly, proteins were extracted by homogenizing thoracic aortic tissues in 500 µL 1×RIPA buffer enriched with protease inhibitors (1 µg/mL phenylmethylsulfonyl fluoride and 1 µg/mL leupeptin ). Proteins were resolved and transferred onto a nitrocellulose membrane using standard procedures. The membranes were incubated for 1 h with blocking buffer, then overnight with primary antibodies against G6PD (Cell Signaling Technology 12263, Danvers, MA, USA, 1:1000), CSRP2 (Abcam ab178695, Cambridge, UK; 1:1000), TUBA4A (Sangon D110022, Shanghai, China, 1:1000) and β-Actin (Santa Cruz Biotechnology sc1616, Santa Cruz, CA, 1:1000) at 4℃ overnight. Finally, the membranes were probed with secondary antibody (1: 5000) at room temperature for 1 h. The membranes were washed thrice, then protein intensities determined using the Odyssey® Imager system (LI-COR, Lincoln, NE, USA).
2.9 Statistical analysis
The GraphPad Prism 6 software (San Diego, CA) was utilized for data analysis. The Student’s t-test was applied in group comparisons. Data are shown as means ± standard deviations (SD). Data followed by p < 0.05 were considered statistically significant.