2.1. Animals and epididymis sample collection
Sample collection was carried out under license in accordance with the Guidelines for Care and Use of Laboratory Animals of China and all protocols were approved by the Institutional Review Board of Southwest University of Science and Technology . Male yaks (Maiwa yaks) (n = 3; age, 1 year; named M1, M2, and M3) and Male cattleyaks (Maiwa yak × Tibetan taurine) (n = 3; age, 1 year; named P1, P2, and P3) were sampled from Maiwa yak population fed on a farm in Hongyuan county, Sichuan province of China.
Epididymis of yaks and cattleyaks were obtained by veterinary surgical operation and fat and connective tissues were removed. Epididymis were then separated apart from testis by fine-scale dissection and were preserved in liquid nitrogen (-196 °C), transported to the laboratory and stored at -80 °C until analysis. The animals would not be killed after sample collections. The animals would rather be kept in a safe and contained place close to the farm-house and away from hazards.
2.2. Extraction of the total protein from epididymis tissue
For protein extraction, each epididymal tissue sample was ground in a mortar with liquid nitrogen; an appropriate amount of lysis buffer (7 M urea, 2 M thiourea, 0.1% CHAPS) was added to the samples and vortexed to mix. The lysates were then extracted with ultrasonication (60 s, 0.2 s on, 2 s off, amplitude 25%), and placed at room temperature for 30 minutes. Subsequently, the lysates were centrifuged at 15000 rpm at 4 ° C for 10 min, after which supernatant was carefully collected and stored at -80 °C. Concentration of the extracted protein was measured using the Bradford method. The sample was diluted with lysis buffer to obtain a final concentration within the range of the standard curve. BSA was dissolved with lysis buffer to a series of standard protein concentrations. A 10 µL diluted sample and standard product were collected separately, and reacted respectively with 300 µL protein quantitation dye under dark for 15 ~ 20 min. Simultaneously, the absorbance was read at 595 nm with a microplate reader (Thermo, model: Multiskan MK3) for the standard product and the sample. The standard curve of the relationship between absorbance and concentration of the standard product in each tube was obtained, and the protein concentration of each sample was calculated.
2.3. Enzymatic digestion of the proteins
For trypsin digestion, a 100 µg of total protein was used by a filter-aided sample preparation protocol (FASP). The samples were reduced with 10 µL of reducing reagent at 37 °C for 1 hour and followed by addition of 2 µL of cysteine-blocking reagent for 30 minutes at room temperature. The reductive alkylated protein solution was then added to a 10 K ultrafiltration tube (Merck Millipore, REF UFC501096), centrifuged at 12,000 rpm for 20 minutes, and bottom solution of the collection tube was discarded. Afterwards, 100 µL dissolution buffer was added in the iTRAQ kit and centrifuged at 12,000 rpm for 20 min. Before collection tube replacement, the bottom solution in the collection tube was discarded, and the process was repeated 3 times. Subsequently, 4 µg (1:50 compared to protein) trypsin (Promega REF V5111) in a volume of 100 µL was added to the ultrafiltration tube and reacted overnight at 37 °C. The next day, the sample was centrifuged at 12,000 rpm for 15 min after which the peptide solution after enzymatic digestion was retained at the bottom of the collection tube. 200 µL of ddH2O was then added to the ultrafiltration tube and centrifuged again at 12,000 rpm for 15 min. Finally, 500 µL of the digested sample was collected from the bottom of the collection tube.
2.4. iTRAQ labeling
For the iTRAQ labeling, the iTRAQ reagent (8-plex; AB Sciex) was removed from the freezer and equilibrated to room temperature. 150 µL isopropanol was added to each tube of iTRAQ reagent, vortexed and centrifuged to the bottom of the tube. Thereafter, 50 µL of sample (100 µg of enzymatic product) was transferred to a new centrifuge tube, iTRAQ reagent was added to the sample, and then vortexed, centrifuged and reacted at room temperature for 2 hours. 100 µL of ddH2O was added to the mixture to stop the reaction.
2.5. Offline pre-separation of enzymatically digested peptides and LC-MS/MS mass spectrometry
The samples, after the labeled lyophilized peptides, were dissolved in 100 µL mobile phase A [98% ddH2O, 2% acetonitrile (pH 10) (Merck, Cat. No: 100030, Germany)], centrifuged at 14,000 rpm for 10 min, and the supernatant was taken for use. 200 µg enzymatically decomposed bovine serum albumin (BSA, Sigma-Aldrich, Cat. No. A2058, USA) was added to the column (Durashell-C18, 4.6 mm × 250 mm, 5 µm, 100 Å) (Agela, Cat. No. DC952505-0) to test the conditions of isolation (column temperature: 45 °C, detection wavelength: 214 nm). Thereafter, 100 µL of the prepared sample was loaded at a flow rate of 0.7 mL/minute. The separation gradient was as follows: 0 min: 5% mobile phase B (98 % acetonitrile, 2% ddH2O) (ddH2O adjusted the pH to 10 with ammonia); 5 min: 8% mobile phase B; 35 min: 18% mobile phase B; 62 min: 32% mobile phase B; 64 min: 95% mobile phase B; 68 min: 95% mobile phase B; and 72 min: 5% mobile phase B . For protein analysis, the nanoscale reversed-phase chromatography connected to mass spectrometer (Thermo, Model: Fusion) was used. The segments obtained by high pH reversed-phase were reconstituted with 20 µL (2% methanol, Sigma-Aldrich, article number: 14262, USA), (0.1% formic acid, Sigma-Aldrich, Cat. No. 56302, USA), and then centrifuged at 13,000 rpm for 10 min. Thereafter, a 10 µL volume was loaded by a sandwich method with a loading pump flow rate 350 nL/min over 15 min. The isolation flow was 350 nl/min and isolation gradient was as follows: 0 min: 4% mobile phase B (100 % acetonitrile, 0.1 % formic acid); 5 min: 15% mobile phase B; 40 min: 25% mobile phase B; 65 min: 35% mobile phase B; 70 min: 95% mobile phase B; 82 min: 95% mobile phase B; 85 min: 4% mobile phase B; and 90 min: 4% mobile phase B . The mass spectroscopy (MS) parameter settings were as follows: spray voltage 2.1 kV; capillary temperature: 250 °C, and Scan range: 350-1800m/z.
2.6. Data analysis
The selection of the database was based on the species required, the completeness of database annotations and the reliability of sequence. The used database was bovine protein sequence library, and the mass spectrometry analysis of iTRAQ was performed by Fusion mass spectrometry. The original mass spectrometry files were processed by Thermo's commercial software Proteome Discoverer 1.4. The search parameters were set as follows: enzyme, trypsin; static modification, C carboxyamidomethylation (57.021 Da); dynamic modification: M oxidation (15.995 Da), N terminal; species, Acyrthosiphon pisum; precursor ion mass tolerance, ± 15 ppm; fragment ion mass tolerance, ± 20 mmu; and max missed cleavages, 2.
The significant differentially abundant proteins (DAPs) were screened by T-test (t-test). Proteins with a p-value less than 0.05 and a fold difference greater than 1.5 or less than 0.67 were considered DAPs.
2.7. GO, KEGG and PPI analysis of DAPs
DAPs between yak and cattleyak epididymal tissues were mapped to Gene Ontology (GO) terms in the database (http://www.geneontology.org/). GO is a database established by the Gene Ontology Consortium, which is suitable for all species to define and describe the functions of genes and proteins. The calculated p-value was adjusted through Bonferroni Correction, taking enriched p-values as a threshold. Pathway analysis of identified proteins could deepen understanding of the metabolic capacity of the species, biological processes information, and related diseases. KEGG database (Kyoto Encyclopedia of Genes and Genomes database) was used to enrich biological pathways of the DAPs. Using the String Protein Interaction Database, an interactive network analysis of significantly expressed proteins was performed and differential protein interaction network data files were directly imported into Cytoscape software for visual editing.
2.8. Enzyme linked immunosorbent assay (ELISA)
ELISA was carried out to confirm the differential abundances of eight proteins, namely CD63, ELP3, LSM5, GSTM1, GGH, ERAP1, GPX5 and MUC15. Briefly, total protein was taken out of each sample according to the manufacture's protocol of DNA/RNA/protein co-extraction Kit (Tiangen Biotech (Beijing) Co., Ltd., China). Total protein concentration of each sample was detected by NanoDrop 3000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and adjusted to 0.1 mg/mL. ELISA detection was performed according to the manufacturer's instructions (MyBioSource, Inc., San Diego, CA, USA). The first antibodies against the proteins mentioned above were all originated from Bos taurus and all ELISA kits were provided by MyBioSource (San Diego, CA, USA). The standard sample provided by the manufacture was used to establish the standard curve and regression equation. Each sample was analyzed with triplicates and the OD value of each well was determined using microplate reader set to 450 nm within 15 min. The concentrations of the targeted proteins were calculated from the regression equation and the content of each protein was determined from each sample. Statistical analyses were performed using SPSS 22.0 standard version (IBM, Armonk, NY, USA). Student's t-test was employed to analyze differences of each protein expression level between the control (yak) and tested groups (cattleyak). For all tests, statistical significance was taken as p ‹ 0.05.