The Beijing-You (BJY) and Cobb-Vantress (Cobb) eggs were obtained from the experimental farm of the IAS (CAAS, Beijing, China) and the Anhui five-star cultivation group CO. LTD (Ningguo, China). All eggs were incubated with the normal procedure and chicks were reared in caging under continuous lighting using standard conditions of temperature, humidity and ventilation at the farm of the IAS, CAAS. Chickens used for sample collection at D 1 were not fed. The same diet was fed to all chickens and was formulated to be intermediate between recommendations for the two breeds [36, 37]. The starter ration (D 1 to D 21) provided 20% crude protein and 2.87 MCal/kg energy. Feed and water were provided ad libitum.
Breast muscles were collected from embryos at ED 12 and ED 17 or chicks of similar weights at D 1 and D 14 within each breed. Chicks were electrically stunned and killed by exsanguination. Because of the limited amount of sample from each bird, especially during the embryonic stages, 10 embryos or birds with similar egg/body weight were used for protein analysis, an additional 10 for histology and 10 for metabolomic analysis. The embryos, bodies and breast muscles on both sides were weighed. Sub-samples were immediately fixed in 4% paraformaldehyde and held at room temperature and additional samples were snap frozen in liquid nitrogen and held at −80 °C. The latter samples were used for the iTRAQ, UHPLC–MS, Western blotting and oil red O staining.
Two or three serial cross-sections of five randomly selected chickens per age in the two breeds were used to evaluate the area, diameter and density of muscle fibers (stained with hematoxylin and eosin) and oil red O staining. Fixed tissues were dehydrated through an ascending ethanol series, embedded in paraffin, and sectioned (3–5 μm). After dewaxing in xylene and rehydration using a descending alcohol gradient, mounted muscle sections were stained with hematoxylin and eosin (H&E). Oil red O staining was as follows: frozen sections (4–8 μm) were air dried for 15–20 min then immersed in 100% isopropanol for 5 min, 0.5% working solution of oil red O for 7–8 min then 85% isopropanol for 3 min. Thereafter, sections were washed with three exchanges of deionized water and counterstained with hematoxylin for 1–1.5 min to visualize nuclei. Sections were rinsed with running tap water for 10 min and covered with a coverslip using 10% glycerol in PBS. Images were captured and processed with Image-Pro Plus 6.0 software.
Protein extraction and quantitation
The muscle samples obtained at four ages from the two breeds were ground to powder in liquid nitrogen and dissolved in lysis buffer (9 M urea, 4% CHAPS, 1% DTT, 1% IPG buffer) at 30 °C for 1 h. The supernatants, obtained by centrifugation at 15,000 g for 15 min at room temperature, were re-centrifuged. Protein concentrations of the supernatants were then determined by the Bradford method. For each age and breed, two pools were constructed using equal amounts of protein from five of the 10 sampled individuals. Each pool was then diluted to the same concentration with TBS for iTRAQ labeling and then stored at −80 °C until analysis.
Protein digestion and iTRAQ labeling
The two protein pools, representing biological replicates at each stage and breed, were used to determine the protein profiles. After precipitation with acetone, protein pellets were dissolved in the buffer from the iTRAQ kit (Sigma, St Louis, MO). Each sample was then reduced, alkylated, digested with trypsin, and labeled with the iTRAQ reagents as follows: each protein pellet for eight samples was reduced with 4 μL reducing reagent, incubated at 60 °C for 1 h, alkylated with 2 μL cysteine blocking reagent, and incubated at room temperature for 10 min. Trypsin digestion used 2.5 μg sequencing-grade trypsin (Promega, Madison, WI) and incubating the samples at 37 °C for 12 h. The peptides were precipitated and collected by centrifugation at 15,000 g for 20 min, then dissolved in 50 μL dissolution buffer and re-centrifuged.
The labeling was 2-plex for the two pools at each age and breed. For BJY, reporter tags 117/118, 115/116, 113/114, 119/121 were used for samples from ED 12, ED 17, D 1 and D 14, respectively. For the Cobbs, iTRAQ labeling used 115/116, 113/114, 119/121, 117/118 for samples from ED 12, ED 17, D 1 and D 14, respectively. The labeled peptide samples were pooled within each breed (8-plex, 100 g total peptide) and fractionated by strong cationic exchange (SCX) chromatography.
Chromatographic separation of the pooled samples was performed on an Agilent 1200 HPLC system (Agilent Technologies Inc., Santa Clara, CA). Labeled peptides were fractionated by strong cation exchange liquid chromatograph (SCX) using a 2.0 × 150 mm, 5 μm, 300 Å column (Michrom, Auburn, CA). The samples were dissolved in 100 μL buffer A (10 mM ammonium formate (pH = 2.8) with 20% ACN). Separation was performed at 0.3 ml/min using a nonlinear binary gradient from buffer A to 50–100% buffer B (500 mM ammonium formate (pH = 2.8) with 20% ACN) over 40 min, and 30 to 100% buffer B for 5 min. A total of 12 fractions were collected. The first fraction was collected from 0 to 5 min, and then 4 min fractions were collected from 6 to 44 min, and a final fraction from 45 to 50 min. Each fraction was vacuum freeze-dried for LC-MSMS analysis.
Fractions were dissolved in Nano-RPLC Buffer A (0.1% formic acid, 2% ACN). The online Nano-RPLC was employed on the Eksigent nanoLC-Ultra™ 2D System (SCIEX, Framingham, MA). The samples were loaded on a C18 nanoLC trap column (100 μm × 3 cm, C18, 3 μm, 150 Å) and washed with Nano-RPLC Buffer A (0.1% formic acid, 2% ACN) at 2 μL/min for 10 mins. Elution used a gradient of 5% to 35% Nano-RPLC buffer B (0.1% formic acid, 98% ACN) over 70 min using an analytical ChromXP C18 column (75 μm × 15 cm, C18, 3 μm 120 Å) with spray tip. Data was acquired by the Triple TOF 5600 System (SCIEX) fitted with a Nanospray III source (SCIEX) and a pulled quartz tip as the emitter (New Objective, Woburn, MA). The ion spray voltage of the mass spectrometer was set to 2.5 kV, the curtain and nebulizer gases were set to 30 psi and 5 psi, respectively, and the heated capillary temperature was set to 150 °C. Survey scans were acquired in 250 ms using the information dependent acquisition. Up to 35 product ion scans were collected if they exceeded 150 counts per second (counts/s) with a 2+ − 5+ charge-state. The total cycle time was fixed at 2.5 s. For collision-induced dissociation, a rolling collision energy setting was used to all precursor ions. A dynamic exclusion time was set for 1/2 of peak width (18 s). The precursor was then refreshed off the exclusion list.
Differential abundance (DA) protein identification and quantification
The raw peptide, protein identification and quantification were performed using Protein Pilot v4.0 (SCIEX) against the chicken database with the Paragon algorithm . Search parameters were set as follows: instrument was TripleTOF 5600, iTRAQ 8-plex quantification, trypsin as enzyme, fixed modification of cysteine by iodoacetamide, biological modification as the ID focus, thorough identification search. To reduce false positive identification, false discovery rate (FDR) less than 1% was required for all reported proteins by PSPEP (Proteomics System Performance Evaluation Pipeline Software, integrated into the Protein Pilot software). Proteins with > |1.5| -fold differences between coupled samples and P-value of less than 0.05 were determined as having differential abundance.
Metabolomic sample preparation and quantitation
Breast muscle samples from 10 birds in BJY and Cobb at four different ages were prepared by powdering the tissue in liquid nitrogen. Sample (0.1 g) was mixed with 500 μL of 50% aqueous acetonitrile (Merck, Darmstadt, Germany) and centrifugation at 12,000 g and 4 °C for 15 min to eliminate the proteins. Supernatant (200 μL) was removed and diluted with 100 L acetonitrile (Merck, Darmstadt, Germany), then vortexed for 5 min. After centrifuging at 12,000 g and 4 °C for 10 min, the clear supernatant was transferred to the sampling vial and an aliquot of 80 μL was injected for UHPLC–MS analysis.
UHPLC-MS analysis was performed on a 1290 Infinity LC system equipped with 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer (Agilent). Chromatographic separations were performed at 40 °C on a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Milford, MA).The mobile phase consisted of 0.1% formic acid - water (A) and 0.1% formic acid - ACN (B). The optimized elution started from 5% buffer B at 0–2 min and increased to 95% buffer B between 2 and 17 min, then held at 95% buffer B for 2 min, followed by re-equilibrating for 6 min. The flow rate was 0.4 mL/min and the injection volume was 4 μL. The autosampler was maintained at 4 °C.
An electrospray ionization source (ESI) was applied in positive and negative mode. The optimized parameters were set up as follows: capillary voltage, 4 kV for positive mode and 3.5 kV for negative mode; nebulizer pressure, 45 psig (310.3 kPa); drying gas flow, 11 L/min; gas temperature, 350 °C; fragmentor voltage, 120 V; skimmer voltage, 60 V. Data were acquired in centroid mode from 50 to 1100 m/z.
DA metabolite identification
Each sample was represented by a total ion current chromatogram. Agilent MassHunter Qualitative software was applied to convert the UHPLC–MS raw data to common data format files. The program XCMS (http://enigma.lbl.gov/xcms-online/) was used for peak detection, RT alignment and peak integration to generate a visual data matrix. The data of each sample were loaded to SIMCA-P software (MKS Umetrics, Umea, Sweden) for partial least squares-discriminate analysis. Statistically significant differences between mean values of two groups were tested by Student T-test in SPSS 19.0. The differences were considered significant when VIP (Variable Importance in the Projection) value >1 and P < 0.05.
Integrated analysis of DA proteins and metabolites with IPA analysis
Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood City, CA; https://www.qiagenbioinformatics.com/products/ingenuity-pathwayanalysis/) was performed to identify the molecular pathways and network based on DA proteins and metabolites. Metabolite names were converted to the Human Metabolome Database (HMDB) ID with MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/). The list of protein IDs and HMDB IDs was imported into the online software package IPA to determine their canonical pathways and molecular networks. The IPA content version is 27,821,452 and Release Date at 2016–06-14. Analyses were performed with thresholds of P < 0.05; both direct and indirect relationships were considered.
Verification of protein abundance by western blotting
Sliced muscle tissue of each of two pools of five embryos or chicks at each stage was lysed on ice in RIPA lysis buffer with 1 mM PMSF (phenylmethanesulfonyl fluoride, Beyotime, Shanghai, China) and quantified by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Shanghai, China). Samples containing 32 g of total protein were separated by electrophoresis on 12% (w/v) SDS-PAGE and transferred to a PVDF membrane (Millipore, Darmstadt, Germany) for 1.5 h at 200 mA. After blocking for 1 h with 5% (w/v) nonfat dry milk in TBST (0.01% (v/v) Tween 20 in Tris-buffered saline), the membrane was probed with the primary antibodies, indicated below, diluted in blocking buffer, overnight at 4 °C. After washing with TBST and TBS, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG, (CWBIO, Beijing, China), diluted in blocking buffer at room temperature for 1 h. After thorough washing, immunoreactive proteins were visualized by chemiluminescence with images being captured with an ImageQuant LAS 4000 mini machine (GE, Fairfield, CT), and the signal density data were analyzed with Image-Pro Plus 6.0 software. The primary antibodies, all rabbit polyclonals, were raised against: Long-chain specific acyl-CoA dehydrogenase (ACADL, 1:1000, Sigma-Aldrich), ATP synthase subunit beta (ATP5B, 1:3000, Agrisera, Vännäs, Sweden), alpha 2 Macroglobulin (A2M,1:300, Bioss, Beijing, China), Tropomyosin beta chain (TPM2, 1:400, LifeSpan BioSciences, Seattle, WA), Desmin (DES, 1:300, Abcam, Cambridge, MA), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10,000, Abcam), Heat shock protein 70 (Hsp70, 1:2000, Abcam), Pyruvate kinase (PKM2, 1:1000, Thermo Fisher Scientific, Shanghai, China).