Hanwoo cattles were slaughtered at 24 months of age. Bovine longissimus dorsi isolated from the 13th rib were frozen immediately after dissection and stored at −196°C until protein analysis. To isolate protein extracts, bovine longissmus dorsi were weighed and thawed in buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 10 mM DTT). Homogenization (30 mg/ml) was performed at room temperature with a rotor blade homogenizer followed by centrifugation at 15,000 rpm at 4°C for 30 min. The supernatant was aliquoted and stored at −80°C until the 2-DE analysis was performed.

Protein extracts (100 μg) were applied to Immobiline™ Dry strips (pH 3 to 10, 18 cm; Amersham Biosciences, Gjettum, Sweden). Isoelectrofocusing was conducted using Pharmalytes (pH 3 to 10) for a 12-h rehydration period, 1 h in 500 V gradient, 1 h in a 8,000 V gradient, and 13 h at a 8,000-V steady-state level. After isoelectrofocusing in the first dimension, strips were incubated with gentle shaking in an equilibration buffer (50 mM Tris-HCl, pH 8.8; 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue) containing 1% DTT for 15 min, and 2.5% iodoacetamide for 15 min. For the second dimension, the strips were transferred to the tops of 12.5% polyacrylamide gels containing SDS. Proteins were separated for 12 h. After fixation, proteins were detected using a silver staining kit.

Gels were scanned using an ImageScanner flatbed scanner. Protein spots were analyzed using ImageMaster 2-DE Elite software (Amersham Biosciences, Gjettum, Sweden). Spots were detected and quantified automatically. All master images were obtained from 2-DE analysis.

Proteins were subjected to in-gel trypsin digestion. Excised gel spots were destained and incubated with 200 mM ammonium bicarbonate for 20 min. The gel pieces were dehydrated in acetonitrile and dried in a vacuum centrifuge. The dried gel pieces were rehydrated in 50 mM ammonium bicarbonate containing 0.2 μg modified trypsin (Promega, Madison, WI, USA) for 45 min on ice. After removing the solution, 50 mM ammonium bicarbonate was added. A column containing of Poros reverse phase R2 material (20 to 30-μm bead size, PerSeptive Biosystems, Norwlk, CT, USA) was packed in a constricted GELoader tip (Eppendrof, Hamburg, Germany). Thirty L of the peptide mixture from the digestion supernatant were diluted in 5% formic acid, loaded onto the column, and washed with 5% formic acid. For MS/MS analyses, peptides were eluted with 50% methanol, 49% H₂O, and 1% formic acid directly into a pre-coated borosilicate nanoelectrospray needle (Micromass, Manchester, UK). MS/MS analysis of peptides generated by in-gel digestion was performed by nano-ESI on a Q-TOF mass spectrometer (Micromass, Manchester, UK). Product ions were analyzed using an orthogonal TOF analyzer, fitted with a reflector, microchannel plate detector and time-to-digital converter. The data were processed using a Mass Lynx Windows NT PC system (Micromass, Manchester, UK). To identify the proteins, all MS/MS spectra recorded for tryptic peptides recovered from the gel spots were used for searches of protein sequences from the NCBInr databases using the MASCOT program.

Protein extracts (30 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane in 25 mM Tris-HCl buffer (pH 8.8) containing 0.19 M glycine, 0.1% SDS, and 20% methanol at 120 mA for 1 h. The membrane was probed with antibodies specific for myosin light chain 1 (Abcam, Cambridge, UK) and carbonic anhydrase 2 (Novus Biologicals, Littleton, CO, USA) at a 1:500 diultion for 1 h. The blot was then incubated with horseradish peroxidase-labeled anti-rabbit IgG serum for 1 h, and antibody binding was visualized using a SuperSignal enhanced chemiluminescence kit from Amersham Biosciences.

Data for a total of 146 animals from progeny tests at the National Institute of Animal Science, South Korea were used. The animals were ranked based on the MS and divided into two groups: a high MS group (0.4 to 1.5) and low MS group (−0.8 to −0.55). The MS was determined according to the ratio of intramuscular fat content to muscle mass. Differences between these two groups were analyzed with a t-test. A simple correlation analysis was conducted to assess the association between spot intensity and breeding value.

An animal model with BLUP as a mixed linear model was used to estimate the individual breeding values. The animal model used for the analysis was:

in which Y_ijkl is observation _ijkl for the traits, μ_i is the population mean for the ith trait, YS_ij is the fixed effect of jth year-season for the ith trait, L_ik is the fixed effect of kth location of birth for the ith trait, A_ijkl is the random additive genetic effect of individual for the ith trait, b_i is the linear regression coefficient for the ith trait, D_ijkl is the age at slaughter on the day for the ith carcass trait, and e_ijkl is the random residual associated with observation ijkl.

In the present study, we separated bovine samples according to the estimated breeding value of MS. According to the South Korean breeding system established by the National Institute of Animal Science, breeding values for MS between 0.4 and 1.5 were assigned to the high MS group whereas values between −0.8 and −0.55 were assigned to the low MS group (Figure 1). Twenty cows each were placed in the high and low MS groups. Proteins extracted from bovine longissimus dorsi muscle were separated on a 2-DE gel for proteomic analysis. A total of 300 spots were detected in the gel and spot intensities were compared (Figure 1A). The image analysis identified seven spots corresponding to differently expressed proteins (Figure 1B). Among these, four protein spots corresponded to proteins highly expressed in the high MS group compared to the low MS group. The intensity of spot no. 1 was increased 4-times in the high MS group compared to the low MS group. The intensity of spot no. 3 in high MS group was 2-times higher than that of the low MS group (p<0.01). In contrast, the intensity of spot no. 2 decreased in the high MS group compared to the low MS group (p<0.05; Figure 1C). Our results suggest that these proteins in bovine muscle are highly related to MS.

Selected protein spots were excised from the silver-stained gels and subjected to in-gel trypsin digestion. The extracted peptides were analyzed by nano-ESI on a Q-TOF mass spectrometer. Data for the analyzed protein spots in bovine longissimus dorsi muscles are presented in Table 1. Four spots with increased intensities in the high MS group were identified as phosphoglycerate kinase 1, triosephophate isomerase, acidic ribosomal phosphoprotein PO, and capping protein (actin filament) Z-line alpha 2. The three spots with decreased intensities in the high MS groups were identified as 14-3-3 epsilon, carbonic anhydrase II, and myosin light chain 1 (slow-twitch muscle).

All seven spots we analyzed were also significantly correlated with the breeding value of MS. Highly expressed spots in the high MS group were positively correlated with the breeding value for MS. In contrast, proteins with low expression levels in high MS group were negatively associated with the breeding value of MS (Table 1).

In order to validate our proteomic data, we conducted a Western blot analysis for individual tissues from both groups. Since antibodies against myosin light chain 1 and carbonic anhydrase 2 were commercially available, expression patterns of these two proteins in bovine muscle were evaluated. As shown in Figure 2, the level of myosin light chain 1 (slow-twitch muscle) protein tended to decrease in the high MS group compared to the low MS group. Alternatively, expression of carbonic anhydrase 2 was reduced in high MS groups. These results suggest that myosin light chain 1 and carbonic anhydrase 2 may act as putative bioregulator proteins and help improve the MS of bovine sirloin.