Animals and sample collection
The BF and LD were sampled from each animal (two years old, wether, carcass weight of 25-26 kg) as 6 replicate samples, all of which were of Inner Mongolia cashmere goats (Aerbasi, Yiwei White Cashmere Goat Breeding Farm, Erdos, Inner Mongolia). The experiments were approved by the experimental animal ethics committee of Inner Mongolia Agricultural University (GB 14925-2001). And permission were obtained from the farm owner. Animals were slaughtered under controlled conditions after being electrically stunned, and then, the muscles were collected aseptically into enzyme-free tubes, immediately immersed in liquid nitrogen and subsequently stored at -80°C until analysis.
IMF content of meat
The IMF level for the BF and LD was determined using the Soxhlet extraction protocol following the method of Hopkins et al. . Three grams of freeze-dried meat was weighed into a thimble and extracted in 85 mL of hexane for 60 minutes within individual extraction tins. The solvent was then evaporated off for an additional 20 minutes. The tin was then dried for 30 minutes at 105°C to remove any residual solvent. The variation in meat weight before and after extraction was used to calculate IMF content. The final value is expressed as a percentage of meat weight.
Protein extraction and digestion
Each frozen sample was ground to powder in liquid nitrogen and dissolved in lysis buffer (proteinase inhibitors (Roche, Basel, Switzerland), 1% SDS (Coolaber, China)). The samples were then incubated at room temperature for 20 minutes, vortexing for 30 seconds every minute. After 20 minutes of ultrasonication, the samples were centrifuged at 12,000 rpm and 4°C for 30 minutes. The supernatant was collected for further study, and the protein concentration was measured with a bicinchoninic acid (BCA) kit (Tiangen, China).
One hundred micrograms of protein was added to 200 µL of 8 M urea (Sigma, Germany) and 10 mM DL-dithiothreitol (Sigma-Aldrich, Germany) and incubated at 37°C for 1 hour. After centrifugation at 12,000 rpm for 40 minutes, 200 µL of urea was added to each filtrate tube, which was then agitated. Next, the filtrate tubes were centrifuged twice at 12,000 rpm for 30 minutes each. Then, 200 µL of 50 mM iodoacetamide (Sigma-Aldrich, Germany) was added to each filtrate tube; the reaction was allowed to proceed in the dark for 30 minutes, and then, the liquid was removed. Next, 100 µL of ammonium bicarbonate (Fluka, Germany) was added to each filtrate tube, and the samples were centrifuged at 12,000 rpm for 20 minutes. This step was performed 3 times, and then, the liquid was removed. The samples were incubated overnight with trypsin at 37°C and centrifuged at 12,000 rpm for 30 minutes. Then, 50 µL of ammonium bicarbonate (Fluka, Germany) was added to each filtrate tube. The samples were centrifuged at 12,000 rpm for 30 minutes, and this step was repeated. The filtrate was collected, freeze-dried, and stored at -20°C .
Two methods, namely, information-dependent acquisition (IDA) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), were used to acquire data from separated peptides on the LC-MS/MS system (Sciex, Framingham, MA, USA). Approximately 2 μg of peptides was injected and separated on a C18 HPLC column (75 μm×15 cm). A linear gradient (120 minutes, going from 5 to 80% B at 500 nL/minute) of 0.1% formic acid in water and 0.1% formic acid in acetonitrile was used to separate peptides. The conditions for IDA were as follows: nominal resolving power of 30,000, time-of-flight (TOF)-MS collection from 350 to 1800 m/z and automated collision energy for MS/MS with IDA scanned from 400 to 1800 m/z. The conditions for SWATH-MS were as follows: 150-1200 m/z MS1 mass range, 100-1500 m/z MS2 spectra, and nominal resolving power of 30,000 and 15,000 for MS1 and MS2, respectively.
Protein Pilot 4.5 software (Sciex, Framingham, MA, USA) was used with the UniProt/SWISS-PROT (https://www.UniProt.org/#) database (downloaded from https://www.UniProt.org; 556,388 proteins) to identify peptides. The results were filtered at a 1% FDR. The selected search parameters included the use of trypsin as the enzyme, allowing up to two missed cleavage sites. The peptide mass tolerance was ± 15 ppm, and the fragment mass tolerance was 20 mmu. The data were loaded into PeakView (Sciex, Framingham, MA, USA) software to search the SWATH databank using the ion library generated in Protein Pilot. PeakView generated extracted ion chromatograms (XICs) after processing targeted and nontargeted data. Then, the results were interpreted and quantitatively analyzed using MarkerView software (Sciex, Framingham, MA, USA). MarkerView allows a rapid review of data to determine the DEPs. PCA and volcano plot analysis, which combined fold change analysis and t-tests, were performed. A fold change >2 or fold change < 0.5 and statistical significance (p value < 0.05) were used to identify DEPs .
The DEPs were subjected to bioinformatics analyses. g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) online software was used to perform the GO and KEGG pathway analyses. Cytoscape_v3.6.1 was used to perform the PPI analysis of DEPs, and cytoHubba was used for screening hub genes .
The homogenized denatured protein was separated by SDS-PAGE and transferred in a semidry state to a PVDF membrane (Bio-Rad, USA). The PVDF membrane was blocked in blocking buffer (Li-CoR, USA) for 2 hours and incubated overnight with murine monoclonal primary antibody (Abcam, Germany). The membrane was washed 3 times for 10 minutes each time and then incubated with fluorophore-conjugated goat anti-mouse antibody (Li-CoR, USA) for 1 hour. Finally, the membrane was rinsed with water, and the immunoreactive bands were examined using a LI-COR Odyssey (CLX-0496, USA) near-infrared imager.