Management of experimental animals
A flock of the layer-type L2 strain of TCCs, originally bred for egg production by National Chung Hsing University64, was reared in the university farm and, at the age of 30 weeks, 197 roosters were used in the study. Animal care and use complied with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing University, Taiwan, ROC (IACUC Permit No. 104–112). The roosters were given pellet breeder diet (16.9% crude protein, 3.24% calcium, and 2,930 kcal/kg metabolizable energy) until the end of the experiment. Feed and water were provided ad libitum. Before treatment, the roosters were transferred to individual wire-floored cages in a climate chamber for an adaption period of 2 weeks under the following conditions: a 14:10-h light:dark photoperiod, 25°C, and 55% relative humidity (RH).
Conditions of acute heat stress and sample collection
A total of 192 roosters were treated with acute heat stress at 38°C and 55% RH for 4 h, as described in our previous study65. Five roosters were kept at 25°C and 55% RH as a control group throughout the experiment. Individual body temperature was measured by inserting an alcohol thermometer approximately 2.5 cm into the cloaca before heat treatment and at 0.5, 1, 2, 3, and 4 h into heat treatment. Blood samples were collected from the jugular vein before and after acute heat stress, and plasma was isolated and stored at −80°C until hormone analysis. The roosters were grouped by difference in body temperature between the highest value during acute heat stress and value before heat stress. This grouping resulted in definition of a resistant group (ΔT ≤ 2.5°C) and a susceptible group (ΔT ≥6.5°C). The control group and five roosters from each of the heat-stressed groups were sacrificed for adrenal gland collection for protein expression and histone modification analysis.
Plasma epinephrine and corticosterone analysis
Plasma epinephrine and corticosterone (CORT) levels were measured using Adrenaline Research ELISA (BA E-5100, ImmuSmol SAS, Bordeaux, France) and the Corticosterone ELISA Kit (501320, Cayman Chemical, Ann Arbor, MI, USA), respectively.
Protein sample preparation, isobaric tags for relative and absolute quantitation (iTRAQ) analysis, and fractionation of peptides
The collected adrenal glands were sliced into small pieces and lysed in O’Farrell’s lysis buffer (9.5 M urea, 65 mM dithiothreitol, 2% v/v Ampholyte 3-10, and 2% NP-40). The samples were sonicated (80 W; four times for 10 s) to dissolute proteins. The homogenates were maintained at 4°C for 1 h and centrifuged at 14,000 ´g at 4°C for 10 min to obtain supernatants. The supernatants were mixed with 100% trichloroacetic acid (TCA) to obtain a final TCA concentration of 20% and maintained at 4°C for 1 h with shaking every 15 min. After centrifugation at 14,000g at 4°C for 10 min, the precipitated pellets were collected and washed with ice-cold acetone twice. The protein pellets were air-dried for 10 min and dissolved in 4 M urea solution. Protein concentrations were determined using the Bradford method with bovine serum albumin as the standard66.
This study performed iTRAQ labeling according to the manufacturer’s protocol (iTRAQ reagent multiplex kit, Applied Biosystems, Waltham, MA, USA). Five replicated protein samples from the same group were mixed and used for reduction and alkylation, which was followed by overnight digestion with trypsin. The tryptic peptides from the control, resistant, and susceptible groups were labeled with isobaric iTRAQ tags with mass 114, 115, and 116 Da, respectively. The samples were then pooled, dried using a SpeedVac evaporator (Tokyo Rikakikai Co. Ltd., Bunkyo-ku, Tokyo, Japan), and stored at −80°C until analysis.
Fractionation of the labeled peptides was performed using an ultraperformance liquid chromatography (UPLC) system (ACQUITY UPLC System, Waters, Milford, MA, USA) and a 2.1 mm × 150 mm × 1.7 µm column with a volume of 0.519 mL (ACQUITY UPLC BEH C18, Waters). The mobile phase was prepared in a gradient with 10 mM ammonium bicarbonate (ABC, pH 10, mobile phase A) and 10 mM ABC/90% acetonitrile (pH 10, mobile phase B). A gradient was created with mobile phase B from 0% to 3% during min 0–5; 3% to 30% during min 5–40; 30% to 70% during min 40–55; and 70% to 0% during min 55–60. The flow rate was 0.2 µL/min. Fractions were collected in 1-min intervals for 1 h duration. Urea solutions in various fractions were removed using C18 ZipTip (Merck, Darmstadt, Germany). All fractions were dried using a SpeedVac evaporator (Tokyo Rikakikai Co. Ltd.) and stored at −80°C until analysis.
Protein identification using nano-UPLC–electrospray ionization (ESI)–quadruple time-of-flight (Q-TOF)–MS/MS
A nano-LC-MS/MS system was used to analyze the tryptic peptides. The peptides were separated using an Ultimate 3000 LC RSLC nano-LC system (Dionex-Thermo Scientific, Chelmsford, MA, USA) coupled with a Q-TOF mass spectrometer (maXis impact, Bruker Daltonics Inc., Bremen, Germany). Each dried fraction was dissolved in 10 µL loading buffer (2% acetonitrile (ACN) and 0.1% FA) and injected into a C18 trapping column (Acclaim PepMap C18, Dionex-Thermo Scientific) connected to a C18 analyst column (Acclaim PepMap C18, Dionex-Thermo Scientific) for peptide separation. The labeled peptides were eluted using a linear gradient of mobile phase A (2% ACN and 0.1% FA) and mobile phase B (80% ACN and 0.1% FA) applied at a flow rate of 0.3 µL/min for 90 min. The gradient conditions were as follows: 5% to 30% mobile phase B during min 5–65; 30% to 98% mobile phase B during min 65–79, and finally, down to 10% mobile phase B within 1 min.
The mass spectrometer was operated at 50–2000 m/z at 2 Hz, and the 20 most intense ions with 420–2000 m/z in each survey scan were selected for the MS/MS experiment. MS/MS data were acquired from 50 to 2000 m/z at 5–10 Hz. The MS/MS spectra were de novo sequenced and assigned a protein ID by using PEAKS X (Bioinformatics Solutions, Waterloo, Canada) and searched against the NCBInr database (NCBInr 20180904 version) for protein identification. Protein quantification was achieved using PEAKS X with significant score (−10logP) > 15, and at least one unique peptide was detected. Proteins quantified using iTRAQ as having a 1.3-fold change for a high (>1.3) or low (<0.77) level of relative abundance were considered to be differentially expressed proteins (DEPs).
Bioinformatics analysis of DEPs
The DEPs among the groups were annotated for their cellular components, biological processes, and molecular functions by using the Gene Ontology database (amigo1.geneontology.org/cgi-bin/amigo/go.cgi).
Histone sample preparation, chemical derivatization, trypsin digestion, and desalting
Histones were isolated using a modified protocol67. Briefly, nuclei were isolated with nuclei isolation buffer (NIB; 15 mM Tris, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 250 mM sucrose and protease inhibitor cocktail tablet; pH 7.5) and 0.2% NP-40. After they had been cut into small pieces, the adrenal glands in NIB were homogenized using a homogenizer (T 10 basic ULTRA-TURRAX, IKA, Guangzhou, China), which was followed by 10 min incubation on ice. The mixture was centrifuged at 1,000 ´g at 4°C for 10 min, and the resultant nuclei pellets were collected. The pellets were washed with NIB twice. Histones were then acid-extracted from the isolated nuclei by using 0.2 M H2SO4 at 4°C for 4 h with shaking every 15 min. The histone-containing supernatants were mixed with 100% TCA to a final TCA concentration of 33% and incubated on ice for 1 h. The histone-enriched pellets were washed with ice-cold acetone/0.1% hydrochloric acid and ice-cold acetone and centrifuged to enable pellet collection. The collected pellets were air-dried and reconstituted in double-distilled water. Finally, the histones were purified through centrifugation and quantified for concentration by using the Bradford method with bovine serum albumin as the standard (Peterson, 1983). All samples were dried using a SpeedVac evaporator (Tokyo Rikakikai Co. Ltd.) and dissolved in 40 μL of 50 mM ammonium bicarbonate, which had pH 8 (concentration > 1 μg/μL). Histones were prepared for MS analysis through propionic anhydride chemical derivatization, trypsin digestion, and propionylation of histone peptides at N-termini, as was described by Sidoli et al. (2016). Then, all histone peptides were desalted with C18 ZipTip (Merck), dried using the SpeedVac evaporator, and finally stored at −80°C until analysis.
Identification of histone modifications by using nano-UPLC-ESI-Q-TOF-MS/MS
Nano-LC-MS/MS and the protocol for identification of histone modifications were performed as is described in section 2.5. Briefly, histone peptides dissolved in 10 µL of loading buffer were separated and eluted using a linear gradient of mobile phase A (2% ACN, 0.1% FA) and mobile phase B (80% ACN, 0.1% FA) applied at a flow rate of 0.3 µL/min for 90 min. The gradient conditions were as follows: 10% to 40% mobile phase B at min 6–74, 40% to 99% mobile phase B at min 74.1–79, and finally, down to 10% mobile phase B within 1 min.
The MS parameters were as described in section 2.5. Label-free quantification was performed using the quantitation module of PEAKS X. Modified histone peptides were identified using PEAKS X through the following search parameters: parent mass error tolerance: 80.0 ppm; fragment mass error tolerance: 0.07 Da; enzyme: trypsin; maximum number of missed cleavages: 2; digestion mode: specific; fixed modifications: propionyl (N-term): 56.0; variable modifications: oxidation (M): 15.99, acetylation (K): 42.01, dimethylation (K): 28.03, methylation (K): 14.02, trimethylation (K): 42.05, propionyl (K): 56.03, deamidation (NQ): 0.98, propionylmethyl: 70.04; maximum number of variable PTMs per peptide: 9; reported number of peptides: 5; and data refine dependencies: 1, 4, 3, 2, 5, 6, 7, 8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 19, 18, and 20. The quantification of histone modification was performed using the PEAKS DB database, which provided an overview of all peptides and histone modifications. The relative abundance of a given PTM resulting from single- or co-occurring PTMs was calculated by dividing its intensity by the sum of intensities for all modified and unmodified peptides sharing the same sequence and without missing values. Therefore, the given PTMs could have only a single datum. The quantification of each peptide of co-occurring PTMs on histone H3 was divided by the quantification of all modified and unmodified peptides to obtain a relative quantification of the histone H3 peptide and the crosstalk of PTMs on histone H3.
The concentrations of plasma epinephrine and CORT were analyzed using Student’s t test in the Statistical Analysis System (SAS) software68. The normality of the body temperature changes and relative values of DEPs and HPTMs were assessed using the normality test. Normally distributed data were analyzed using the least squares means procedure, whereas nonnormally distributed data were analyzed using the Kruskal–Wallis test.