Analysis of Longissimus thoracis Protein Expression Associated with

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Analysis of longissimus thoracis protein expression associated with variation in carcass quality grade and marbling of beef cattle raised in the pacific northwestern US. Kara J Thornton, Kalyan C. Chapalamadugu, Eric M. Eldridge, and Gordon K. Murdoch J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02795 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Journal of Agricultural and Food Chemistry

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TITLE AND AUTHORSHIP

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Title. Analysis of longissimus thoracis protein expression associated with variation in carcass

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quality grade and marbling of beef cattle raised in the pacific northwestern US.

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Kara J. Thorntonab, Kalyan C. Chapalamadugub, Eric M. Eldredgeb, Gordon K. Murdochb*

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a

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4815 Old Main Hill, Logan, UT 84322

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b

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Moscow, Idaho 83844-2330

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*Corresponding author: [email protected], 309 Ag/Biotechnology, University of Idaho,

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Current address: Department of Animal, Dairy and Veterinary Sciences, Utah State University,

Department of Animal and Veterinary Sciences. University of Idaho, 606 Rayburn Street

Moscow, Idaho. 83844-2330

1 ACS Paragon Plus Environment


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ABSTRACT AND KEYWORDS

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Longissimus thoracis (LD) samples from 500 cattle were screened for protein expression

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differences relative to carcass quality grade. The LD of the top 5% (low prime and high choice,

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HQ) and bottom 5% (low select, LQ) carcasses were analyzed using two-dimensional difference

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gel electrophoresis and western blot. Following initial screening, 11 candidate proteins were

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selected for Western blot analyses. Differentially expressed proteins were clustered into 4

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groups: (1) heat shock proteins and oxidative protection, (2) sarcomeric proteins: muscle

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maturity and fiber type, (3) metabolism and energetics and (4) miscellaneous proteins. Proteins

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from groups 1 and 2 were greater in HQ carcasses. Alternatively, increased quantities of proteins

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from group 3 were observed in LQ carcasses. Proteomic differences provide insights into

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pathways contributing to carcass quality grade. A deeper understanding of the physiological

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pathways involved in carcass quality grade development may allow producers to employ

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production practices that improve quality grade.

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Keywords. bovine, carcass quality grade, heat shock proteins, proteolysis, proteome


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INTRODUCTION Harvested beef cattle exhibit variation in USDA carcass quality grade, despite being

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raised under similar conditions.1 Marbling of the longissimus thoracis (LD) at the 12th rib and

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degree of maturity are the main factors used to establish carcass quality grade. Carcass quality

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grade is of utmost importance to producers as this value, along with hot carcass weight,

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determines the payout for a carcass. Currently, the underlying molecular mechanisms responsible

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for this variation in carcass quality grade are inadequately understood. Meat quality is a complex

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trait determined by structural and chemical characteristics of the extracellular connective tissue

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network, intramuscular fat deposition, myofiber size and type, and the interactions between the

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heterogeneous populations of cells which comprise skeletal muscle tissue and the substrates that

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they store.2 Consequently, the complexity of both skeletal muscle tissue and the factors which

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determine meat quality lend to a current lack of knowledge regarding the cellular mechanism(s)

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responsible for the observed variation in carcass quality grade. Carcass quality grade is

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correlated with both consumer satisfaction and meat tenderness.3 Inconsistencies in beef carcass

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quality grade are undesirable as there is generally an improved market for higher quality grade

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beef at least up to and including choice.4 Although undesirable, variation in carcass quality grade

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continues to be a problem while causative physiological factors in cattle raised under similar

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management practices are poorly understood.

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Many cellular processes contribute to quality grade of meat products; skeletal muscle

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itself is heterogeneous where proportions of different cell types and their interactions may differ

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within this tissue. Past research relating to beef quality has focused on genomic studies involving

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the use of quantitative real time polymerase chain reaction (qRT-PCR) analyses, microarray, and

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RNA sequencing as well as development of quantitative trait loci (QTL) for genes associated



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with marbling and tenderness in beef.5 However, genomic studies are limited in their capacity to

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elucidate underlying molecular mechanisms responsible for meat quality, as the genome is

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further from the phenotype than the proteome. Therefore, studies analyzing protein expression

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can complement current genomic studies and provide an improved understanding of the complex

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phenotype related to meat quality. The application of proteomics in meat science has been

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employed with the aim of achieving a better understanding of meat quality.6 Previous proteome

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analyses of porcine and beef muscle have focused on differences in meat quality related to

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electrical stimulation,7 post-mortem storage,8 skeletal muscle hypertrophy,9 skeletal muscle

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growth processes,10 finishing diet,11 ageing,7 post-mortem muscle metabolism,12 differences in

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muscle type,13 tenderness differences based on Warner-Bratzler shear force (WBSF) values,14

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accumulation of subcutaneous adipose,15 intramuscular fat content,16 muscle exudate protein17

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and meat color.18 Further, proteomic differences in high and low quality grade beef in Korean

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native cattle have been investigated.19 However, no proteomic screening has compared and

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reported differences in the LD of high and low quality beef based on carcass quality grade alone

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in typical North American cattle breeds.

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This study analyzed differences in the LD of high vs. low quality graded beef carcasses

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using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser

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desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry as a screening tool

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which identified 11 candidate proteins of interest. Candidate proteins were then subsequently

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evaluated and quantified using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

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(PAGE) and Western blot analyses of individual animal samples. The objectives of this study

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was to use an unbiased approach to identify proteins that are differentially expressed in the LD of

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typical North American raised cattle relative to industry accessed carcass quality grade.


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MATERIAL AND METHODS

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Sample Collection

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Samples were obtained from the LD between the 11th and 12th rib. The LD muscle was

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sampled from 500 randomly selected cattle raised in the Pacific Northwestern Unites States and

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Canada and harvested at Washington Beef in Toppenish, WA. All samples were collected on the

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same day. Approximately 24 h after slaughter, carcasses were ribbed and graded, following plant

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protocol. Samples were collected from both steer and heifer carcasses. Approximately 5 g LD

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core samples were obtained within 30 min of exsanguination from the left side of the carcass

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using a homemade coring device. Immediately following collection, samples were snap frozen

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and transported in liquid nitrogen back to Moscow, ID and stored at -80°C until further

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preparation and analysis was performed. The cattle originated from four different feeding

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facilities in the Pacific Northwestern United States and Canada. All cattle were fed following

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typical industry protocols in this area, however no specific diet information is available for these

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animals. Genetic relatedness was not characterized but was not expected to be greater than

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average for a typical mixed cattle operation in the US.

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Determination of Samples for Analyses

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Carcass and quality grade data, determined by camera, of the sampled cattle were

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received from Washington Beef. The top 5% carcass quality grade high (low prime, high choice;

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HQ) and the bottom 5%, carcass quality grade low (low select; LQ) samples were chosen based

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on their carcass quality grade, with marbling score being the value taken most into consideration.

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Carcass grade information was obtained from Washington Beef via the VBG2000 Vision

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Camera. An equal number of steers and heifers were chosen for initial proteomic screening

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analyses; HQ steers (n = 12), LQ steers (n = 12), HQ heifers (n = 12) and LQ heifers (n = 12).



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While marbling score was the parameter taken most into consideration; care was taken to ensure

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yield grades were balanced between the selected groups (p > 0.30) so that yield (lean growth)

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was not the predominant factor in this study. The carcass data of those chosen for analyses can

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be seen in Table 1. Carcasses that graded less than “select” or were “de-railed” during fabrication

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processes were not included in any analyses.

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Protein Extraction

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Frozen LD muscle samples were ground in a super-cooled mortar and pestle under liquid

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nitrogen. Care was taken to minimize subcutaneous adipose tissue contamination in the sample.

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The ground sample consisted of both skeletal muscle and intramuscular adipose tissue. Total

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protein was extracted using a modified protocol.20 In brief, between 50 and 70 mg of ground

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tissue was transferred to 15µL/mg tissue total protein extraction buffer; 50 mM Tris HCl (pH =

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7.52), 150 mM NaCl, 1mM ethylene diamine tetraacetic acid (EDTA), 1% Tergitol, 0.1% SDS

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and 0.5% sodium deoxycholate. Immediately before use, phosphatase and protease inhibitor

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cocktail tablets (Roche, Indianapolis, IN, USA) were added to the extraction buffer. Samples

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were homogenized, using a Retsch Bead Homogenizer MM301 (Retsch, Newtown, PA, USA), 5

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x 20 s at 25 Hz. Following homogenization, samples were placed on a rocking platform for 10

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min at 4°C. All samples were centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was

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removed and stored at -80°C. Total protein was quantified using the Pierce® BSA Protein Assay

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Kit (ThermoScientific, Waltham, MA, USA). A Wallac Victor 2 1420 plate reader (Perkin

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Elmer, Waltham, MA, USA) was used to quantify total protein.

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Pooling of Samples for 2D-DIGE Protein Screening Analysis

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The 2D-DIGE analysis was used as a screening tool to identify candidate proteins that contribute to the variability in carcass quality grade. Samples were pooled into four different


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groups, separated by both sex and carcass quality grade. The four pooled groups were as follows:

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HQ steers, LQ steers, HQ heifers and LQ heifers. Each sample pool consisted of equal amounts

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of protein from each of the 12 animals included in that group. A pool of all 48 samples was also

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created, which was used as an internal standard for each of the 2D-DIGE gels.

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2D-DIGE

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Two 2D-DIGE gels were run, one consisting of pooled samples from the steers and the

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other from the heifers; gels can be seen in Figure 1. Each gel included the pooled samples as well

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as an internal standard consisting of a protein pool from all samples analyzed and was used for

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normalization during analysis. In brief, protein samples were suspended in 2D-DIGE cell lysis

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buffer (30 mM Tris-HCl, pH 8.8, containing 7 M urea, 2 M thiourea and 4% 3-(3-

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cholamidopropyl) dimethylammonio-1-propanesulfonate (CHAPS) (Applied Biomics, Inc.

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Hayward, CA, USA). Protein concentration was measured using the Bio-Rad protein assay

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method (Bio-Rad, Hercules, CA, USA). Each sample was labeled by adding 30 µg of protein and

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mixing with 1.0 µL of diluted CyDye and kept in the dark on ice for 30 min. The labeling

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reaction was stopped by adding 1.0 µL of 10 mM lysine to each sample and incubating in the

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dark on ice for an additional 15 min. The labeled samples were then mixed together. Two times

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2D-DIGE sample buffer (8 M urea, 4% CHAPS, 20 milligrams/milliliter DTT, 2% pharmalytes

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and trace amount of bromophenol blue), 100 uL de-streak solution and rehydration buffer (7 M

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urea, 2 M thiourea, 4% CHAPS, 20 mg/mL DTT, 1% pharmalytes and trace amount of

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bromophenol blue) were added to the labeling mix to bring the total volume to 250 µL. Samples

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were mixed and centrifuged before loading into the strip holder. After loading the labeled

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samples, isoelectric focusing (IEF) (pH 3-10 linear) was run following the protocol provided by

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GE Healthcare (GE Healthcare, Fairfield, CT, USA). Upon finishing the IEF, the immobilized



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pH gradient (IPG) strips were incubated in freshly made equilibration buffer-1 (50 mM Tris-HCl,

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pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 10

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milligrams/milliliter DTT) for 15 min with gentle shaking. The strips were rinsed in freshly

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made equilibration buffer-2 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2%

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SDS, trace amount of bromophenol blue and 45 mg/mL iodoacetamide) for 10 min with gentle

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shaking. Next the IPG strips were rinsed in the SDS-gel running buffer before transferring onto

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12% SDS-gels. The SDS-gels were run at 15oC until the dye front ran out of the gels. Gel images

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were scanned immediately following the SDS-PAGE using Typhoon TRIO (GE Healthcare).

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The scanned images were analyzed by Image Quant software (version 6.0, GE Healthcare),

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followed by in-gel analysis using DeCyder software version 6.5 (GE Healthcare). The fold

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change of the protein expression levels was obtained from in-gel DeCyder analysis (Applied

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Biomics).

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Mass Spectrometry

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Mass spectrometry for spot identification was performed by Applied Biomics (Applied

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Biomics). Spots that showed at least a 1.25 fold difference between high and low quality grade

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groups in either the heifer or the steer samples were picked up by Ettan Spot Picker (GE

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Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel

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spots were washed, and digested in-gel with modified porcine trypsin protease (Promega,

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Madison, WI, USA). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore).

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Peptides were eluted from the Zip-tip with 0.5 µL of matrix solution (α-cyano-4-

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hydroxycinnamic acid (5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25 mM

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ammonium bicarbonate) and spotted on a MALDI plate. MALDI- TOF MS and TOF/TOF

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tandem MS/MS were performed on AB SCIEX TOF/TOF™ 5800 System (AB SCIEX,


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Framingham, MA, USA). MALDI-TOF mass spectra were acquired in reflectron positive ion

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mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra

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were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each

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of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and

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other known background ions). Both of the resulting peptide mass and the associated

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fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT

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search engine (Matrix science) to search the database of National Center for Biotechnology

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Information non-redundant (NCBInr). Searches were performed without constraining protein

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molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and

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oxidation of methionine residues, and with one missed cleavage also allowed in the search

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parameters. Candidates with either; protein score confidence interval % (C.I.) or ion C.I.%

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greater than 95 were considered significant. Candidates with a protein score C.I.% less than 95

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were not included in the results.

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Western Blotting

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Western blot analysis on individual animals was completed for 11 differentially

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expressed proteins according to the 2D-DIGE screening. All Western blot analysis was

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performed on individual animal samples to verify the differences identified in the “pooled” 2D-

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DIGE. Specifications regarding the antibodies are presented in Table 1S. Ten individual samples

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were run on each gel for each protein analyzed. A total of 5 gels for each of the 11 proteins

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analyzed were utilized to analyze individual protein expression for each of the 11 proteins

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analyzed via Western blot analysis. Protein samples were added to Laemmli sample buffer (Bio-

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Rad, Hercules, CA, USA) at a ratio of 1:1 and heated at 95°C for 7 min prior to separation.

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Proteins were separated using SDS-10% polyacrylamide gels. Electrophoresis was performed



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at120 V for 1 h at 4°C using a Bio-Rad Mini PROTEAN tetra cell gel box in running buffer

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containing 25 mM Tris/ 192 mM glycine/ 0.1% (w/v) SDS. The proteins were then transferred to

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an immobilidon-polyvinylidene fluoride (PVDF) membrane using a Bio-Rad trans-blot apparatus

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with a transfer buffer consisting of 20% (w/v) methanol/ 25 mM Tris/192mM glycine/0.05 (w/v)

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SDS. Samples were transferred at 4°C for 90 min at 100 V. After transfer, membranes were

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stained with Ponceau-S (Sigma, St. Louis, MO, USA) for 30 min according to the

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manufacturer’s protocol. Images of Ponceau-S stained membranes were taken using a Fluor-S

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Multi Imager (Bio-Rad, Hercules, CA, USA). Ponceau-S stain was removed as per the

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manufacturer’s protocol. Membranes were then blocked in a 5% (w/v) non-fat milk (NFM) and

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Tris buffered saline (100 mM Tris-HCl, 0.13 M NaCl and 0.0027 M KCl) (TBS) solution for 45

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min at room temperature. Membranes were incubated with primary antibodies in 5% NFM-TBS

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solution overnight at 4°C, at the reported primary antibody dilutions (Table 1S). Membranes

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were washed 3 x 10 min each in TBS with 0.05% Tween 20 (TBST) followed by incubation with

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secondary antibodies for 2 h at room temperature, three separate secondary antibodies

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conjugated with horseradish peroxidase were used; mouse, goat and rabbit (Pierce, Waltham,

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MA, USA) (Table 1S). Membranes were again washed 4 x 5 min each in TBST at room

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temperature followed by a wash in TBS for 5 min. Western Lightning ECL substrate

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(PerkinElmer, Waltham, MA, USA) was used to detect the peroxidase and images were

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developed using a Kodax X-OMAT 100A processor (Kodak, Rochester, NY, USA). Radiograph

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images were taken using a Fluor-S multi-imager (Bio-Rad, USA). Relative densitometric values

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were determined using Quantity One software (Bio-Rad, USA). Western blot data were

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normalized in 2 ways; (1) equal amounts of protein were loaded to each well (2) relative

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densitometric values were normalized to a common band (~45kDa) in the Ponceau-S stained


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image via a ratio. Western blots were run in duplicate; a coefficient of variation of less than 8%

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between gels was considered acceptable to ensure there was limited variation between duplicate

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gels.

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Statistical Analysis

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Fold change differences in the 2D-DIGE gels were compared using DeCyder software.

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High and low quality carcass samples were considered to be differentially expressed at a fold

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change value of at least 1.25 fold in either the heifer or the steer samples. This fold change value

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was adopted to ensure detection of the proteins that were expressed differentially by at least

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25%. All data from 2D-DIGE analyses compared differences between pooled samples. Samples

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of high and low carcass quality grade were compared in both the steers and the heifers, however

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there were no comparisons made between the two sexes. The PROC mixed function of SAS was

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used to statistically analyze the Western blot data; quality grade was included as a fixed variable

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and individual animal was included as a random variable (Statistical Analysis Software version

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9.2, Cary, NC, USA). Statistical analyses were performed on samples with an N=24 and an

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N=48. These two analyses were performed because in the N=24 analyses, the samples from the

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two groups are more divergent from one another in carcass quality grade as they represent the

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more extreme samples (either HQ or LQ) from the random population average. Whereas in the

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other analysis (N=48) the samples are less divergent in carcass quality but the analyses will have

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more power due to greater sample size. P-values ≤ 0.05 were considered statistically significant.

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P-values ≤ 0.10 were considered trends in the data.

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RESULTS

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2D-DIGE Proteomic Analysis



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Proteomic screening of the LD was performed on four different groups of pooled

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samples; HQ steers, LQ steers, HQ heifers and LQ heifers. Proteomes of high and low carcass

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quality graded animals were compared within sexes. The 2D-DIGE analysis utilized pooled

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samples as it served only as a screening tool to determine candidate proteins for Western blot

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analysis of expression differences in individual samples. Validation using Western blot analysis

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of individual samples confirmed many of the differences observed in the 2D-DIGE proteomic

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analyses.

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Seventy-seven distinct protein spots, representing 45 unique proteins were found to be

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differentially expressed in the LD of either or both of the pooled heifer and steer samples (Table

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2). Subsequently, these proteins were separated for simplification of interpretation into four

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groups based upon known functions: (1) heat shock proteins and proteins involved in oxidative

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protection, (2) sarcomeric proteins: muscle maturity and fiber type, (3) proteins of importance in

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metabolism and energetics and (4) miscellaneous proteins.

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Western blot analyses of proteins from the heat shock (HSP) and oxidative protection

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classification revealed that the LD from both LQ steer and heifer carcasses contained

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significantly more heat shock protein beta-1 (HSPB1), alpha-crystallin B chain (CRYAB) and

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protein DJ-1 (PARK7).

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In the sarcomeric proteins related to both muscle maturity and fiber type, proteins that

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regulate structure and function of the contractile apparatus were differentially expressed between

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HQ and LQ carcass LD muscle in both the steers and the heifers. The HQ steers expressed more

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myosin heavy chain 1 (MYH1), α-actin, myosin light chain 1/3, myosin light chain 6B,

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tropomyosin (TPM), myosin regulatory light chain 2 and myosin light chain 1 when compared to

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the LQ steer LD samples. However, expression of isoforms of myosin-binding protein C, myosin


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light chain 1/3, and myosin regulatory light chain 2 were expressed significantly higher in the

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LD of LQ steer carcasses as compared to HQ steer carcasses. The HQ heifers expressed

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significantly more of at least one of the identified isoforms of MYH1, myosin regulatory light

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chain 2 and myosin light chain 1 when compared to LQ heifer carcasses. One of the isoforms of

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myosin-binding protein C, alpha actin, TPM, troponin T, myosin light chain 1/3, and myosin

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regulatory light chain 2 were expressed significantly more in the LD of LQ heifers as compared

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to HQ heifers.

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Our 2D-DIGE/MS proteomic analysis revealed a number of differentially expressed

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proteins between both the HQ and LQ carcass quality graded steers and heifers that are known to

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function in the metabolism and energetics of skeletal muscle. Most of the identified proteins

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were expressed in greater quantity in the HQ samples than LQ samples in both the heifer and

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steer carcasses. However, the LQ carcasses expressed greater amounts of adenosine

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monophosphate deaminase 1 (AMPD1), phosphoglucomutase1, aldehyde dehydrogenase 1,

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carbonic anhydrase 3 and fatty acid binding protein 3 (FABP3) as compared to the LQ steer

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carcasses. All other proteins identified by 2D-DIGE proteomic analysis to be significantly

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different between HQ and LQ steers grouped as being involved in metabolism and energetic of

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skeletal muscle demonstrated increased expression in the HQ carcasses. Similar to what was

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found in the steers, differential expression was greater in the LD of the HQ heifers in proteins

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grouped as being involved in metabolism and skeletal muscle energetics.

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The last group of proteins identified to be differentially expressed in the LD between high

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and low quality carcasses was categorized as “miscellaneous” representing all other proteins

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outside of the other three categories. The proteins serotransferrin precursor, and majority of the

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identified isoforms of myoglobin and hemoglobin were found to be significantly more abundant



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in HQ steer carcasses as compared to LQ steer carcasses. However, albumin and a few isoforms

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of the myoglobin protein were in greater abundance in LQ steer carcasses. The HQ heifer

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carcasses expressed proportionally more albumin, and most of the isoforms of myoglobin and

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hemoglobin when compared to the LQ heifer carcasses. The serotransferrin precursor and a few

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of the isoforms of both hemoglobin and myoglobin were expressed in greater abundance in the

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LQ heifer carcasses as compared to the HQ heifer carcasses.

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Proteins validated with Western blot analysis were chosen based on two different criteria:

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(1) those that exhibited the largest fold change values after 2D-DIGE analysis of pooled samples

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(2) those perceived as having the most physiological relevance to differences in carcass quality

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grade based upon known function.

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Western Blot Analysis

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Subsequent quantification of 11 proteins identified by the 2D-DIGE proteomic and MS

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screening to be differentially expressed in the LD between the HQ and LQ samples in either the

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steers or the heifers was performed using antibody specific Western blotting (Figure 2). In both

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the steers and the heifers, densitometric values were analyzed with an N=24 and an N=48 to

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determine differences in samples more extreme in their quality grade. Analysis of an N=24

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demonstrated that HQ heifer carcasses expressed more (p = 0.03) AMPD1 and less (p = 0.04)

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sarcomeric mitochondrial creatine kinase (SMTCK) when compared to LQ heifer carcasses

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(Table 3). When an N=48 was analyzed similar results were observed. When compared to LQ

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heifer carcasses, the HQ heifer carcasses expressed more AMPD1 (p = 0.005) and less TPM (p =

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0.05) and SMTCK (p = 0.01). Results of Western blot analyses validated differences observed in

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2D-DIGE/MS analysis in 2 and 3 of the proteins at N=24 and N=48, respectively, in samples

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obtained from heifers.


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Western blot analysis of N=24 and N=48 LD samples of HQ vs. LQ steer carcasses

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validated 7 of the differentially expressed proteins identified through 2D-DIGE/MS analysis

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(Figure 3). In an N=24 HQ steer carcasses expressed more AMPD1 (p = 0.05) and HSPB1 (p =

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0.03) as well as less beta enolase (ENO3) (p = 0.006), CA3 (p = 0.02), GPD1 (p = 0.04), TPI (p

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= 0.01) and TPM (p = 0.02) when compared with LQ steer carcasses. The HQ steer carcasses

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also tended to express low quantities of MYH1 (p = 0.09) and PARK7 (p = 0.08) than LQ steer

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carcasses. In an N=48, HQ steer carcasses expressed lower amounts of CA3 (p = 0.0002), ENO3

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(p < 0.0001), GPD1 (p = 0.04), PARK7 (p = 0.05), TPI (p = 0.001) and TPM (p = 0.0003) than

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LQ steer carcasses. LD samples from HQ steer carcasses also tended to have lower quantities of

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SMTCK (p = 0.10), FABP3 (p = 0.06), and MYH1 (p = 0.08). However, the expression of

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FABP3 and MYH1 did not meet the confidence level to confirm differences ascertained by 2D-

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DIGE/MS analyses of the pooled sample sets.

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DISCUSSION

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LD samples represent a heterogeneous cell population that is present within skeletal

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muscle tissue comprised of 3 main cell types: skeletal muscle cells, intramuscular adipocytes and

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fibroblasts. As USDA carcass quality grade/intramuscular fat content was the main factor used to

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determine sample grouping as high vs. low quality carcass merit, the proportions of different cell

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types vary in these samples. For instance, samples from high quality carcasses presumably have

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more mature adipocytes than low quality carcass samples as higher quality grade meat has more

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intramuscular fat. However the marketable product; meat, embodies this complexity and

320

therefore we believe that this total proteomic approach is more advantageous than

321

disadvantageous. Our proteomic and Western blot analyses represents a snapshot of the tissue as

322

a whole, and provides the opportunity to identify some of the underlying physiological



Page 16 of 36

323

mechanisms that may explain meat quality regardless of which cell type(s) are responsible. In

324

fact, this study identified several proteins that are divergently expressed, in HQ vs LQ steers and

325

heifers.

326

HSPs and oxidative protection

327

The present study found that PARK7, a cytosolic chaperone protein that protects against

328

oxidative stress and cell death,21 exhibited increased expression in the LD muscle of LQ steers as

329

compared to LQ steers. PARK7 was not differentially expressed in the LD samples from heifers.

330

To date, no previous studies have analyzed the effects of oxidation in samples obtained from

331

carcasses with divergent carcass quality grade as the main factor. However, it is accepted that

332

oxidative stress is an important process that occurs during ageing and has known impacts on end-

333

product quality. Herein, we report that these same oxidative processes may play a role in

334

determination of carcass quality grade.

335

HSP are essential for normal cell function, and play a role in stress resistance.22, 23

336

HSPB1 exhibited higher expression in the LD of HQ steers when compared to LQ steers. There

337

were no significant differences in HSPB1 expression observed in the heifer samples. To date, no

338

other studies have analyzed the expression of small HSP in relation to carcass quality grade. It

339

has been reported that HSPB1 expression is negatively correlated with intramuscular fat content

340

in the LD of Korean cattle.24 The results of the previous study do not agree with the findings of

341

the present study and likely reflect intrinsic breed and production differences. The HQ samples

342

analyzed in the present study had a significantly higher marbling score when compared to the LQ

343

samples, indicating there was more intramuscular fat in the HQ samples than the LQ samples.

344

Additional research will elucidate the mechanism through which proteins involved in oxidative


Page 17 of 36


345

processes and stress affect carcass quality grade and the authors believe that this represents an

346

exciting new opportunity supported by our proteomic observations.

347

Sarcomeric proteins: muscle maturity and fiber type

348

Our results indicate that both MYH1 and TPM are expressed at higher quantities in the

349

LD of LQ steer as compared to HQ steers. Furthermore, LD samples from heifers showed the

350

same result in TPM expression but not MYH1 expression. These data indicate that there is a

351

greater quantity of proteins that function in muscle maturity and fiber type determination in the

352

HQ carcass samples at the time of harvest, although there was no difference in yield grade

353

between HQ and LQ carcasses. The heterogeneous populations of cells within the samples may

354

have dramatically influenced the expression of these proteins as the HQ carcass samples likely

355

have more intramuscular adipocytes whereas LQ carcass samples likely consisted of fewer

356

adipocytes and more muscle cells which may have skewed the quantifications of these proteins.

357

This explanation is supported by our observation that LQ carcass samples within both the steers

358

and heifers expressed more TPM and MYH1 in Western blot analysis. However, to date, no

359

studies have analyzed proteomic differences in samples obtained from carcasses divergent in

360

quality grade. Future research will determine how distribution of sarcomeric proteins at the time

361

of harvest may impact carcass quality grade.

362

Metabolism and energetics

363

We report that AMPD1 protein is expressed in greater amounts in HQ steers and heifers

364

as compared to LQ steers and heifers. This is the first such report linking the expression of this

365

protein to the protein expression in the LD muscle in cattle based on carcass quality grade. This

366

may suggest that HQ animals have improved energy utilization with respect to adenosine

367

monophosphate metabolism through conversion to inositol monophosphate.



Page 18 of 36

368

In addition, the present study found that CA3, ENO3, GPD1 and TPI have greater

369

expression in the LQ steer samples as compared to the HQ steer LD samples. In addition, all 4 of

370

these proteins showed similar trends in increased expression in the LQ heifers when compared to

371

the HQ heifers, but these data were not significantly different. However, SMTCK was expressed

372

at a significantly higher level in HQ heifers as compared to LQ heifers. SMTCK expression

373

exhibited the same trend in HQ steer samples but only demonstrated a tendency towards

374

significance. To our knowledge, no other studies have analyzed these proteins immediately post-

375

harvest; however many other studies have analyzed changes in the abundance of these proteins

376

during the post-mortem ageing process. Further, several studies have suggested that an increase

377

in glycolytic enzymes, such as ENO3, GPD1, SMTCK, and TPI, may enhance tenderness of the

378

end product.12, 25, 26 However, tenderness was not measured in the present study and as such, we

379

can only hypothesize how these findings may relate to improvements in carcass quality grade

380

and tenderness.

381

Summary

382

Beef quality is a complex trait that involves the integration of many different factors

383

including the structure of the muscle, metabolism within the muscle and intramuscular fat

384

deposition. Proteomic studies are an effective tool to study differential expression of many

385

different proteins, in an unbiased manner that may govern meat quality. This may facilitate the

386

elucidation of the complex mechanisms involved in determining carcass and meat quality.

387

Differential expression of the LD proteome was examined in beef cattle with differing

388

carcass quality grades. 2D-DIGE proteomic techniques were utilized to identify differentially

389

expressed candidate proteins. Western blot analyses on individual samples indicate protein

390

expression differences in HSPs and oxidative stress proteins, sarcomeric proteins involved in


Page 19 of 36


391

muscle maturity and fiber type, proteins involved in the metabolism and energetics of muscle as

392

well as some proteins potentially related to vascularization. Expression of both small HSP and

393

proteins involved in oxidative stress have been reported to influence end-product eating quality,

394

however, this is the first study that demonstrates that expression of these proteins is also related

395

to carcass quality grade. Additionally, many metabolic enzymes and stress proteins increase in

396

skeletal muscle after slaughter, however this process depends upon the myofibrillar proteins

397

present as well as fiber type proportions within skeletal muscle.12 This study confirms that

398

despite being raised under similar conditions, cattle exhibit differences in their quality grade.

399

Some of these differences may be due to differential proteome expression. Beef cattle may have

400

a unique skeletal muscle proteomic “fingerprint” that plays a role in the determination of their

401

quality grade, and thus ultimately impacts both producers and consumers.

402

The proteomic differences we report provide researchers with greater insight into the

403

molecular pathways contributing to carcass quality grade and marbling.

404

ABBREVIATIONS USED

405

LD – longissimus thoracis

406

LQ – low quality carcass

407

HQ – high quality carcass

408

qRT-PCR - quantitative real time polymerase chain reaction

409

QTL – quantitative trait loci

410

WBSF – Warner-Bratzler shear force

411

2D-DIGE - two-dimensional difference gel electrophoresis

412

MALDI – matrix-assisted laser desorption/ionization

413

TOF – time-of-flight



Page 20 of 36

414

SDS – sodium dodecyl sulfate

415

PAGE – polyacrylamide gel electrophoresis

416

EDTA - ethylenediaminetetraacetic

417

CHAPS - 3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate

418

IEF - isoelectric focusing

419

IPG - immobilized pH gradient

420

PVDF - immobilidon-poly-vinylidene fluoride

421

TBS - Tris buffered saline

422

NFM – nonfat milk

423

TBST - TBS with 0.05% Tween 20

424

HSP – heat shock protein

425

HSPB1 - heat shock protein beta-1

426

CRYAB – alpha-crystallin B chain

427

PARK7 – protein DJ-1

428

MYH! – myosin heavy chain 1

429

TPM – tropomyosin

430

AMPD1 - adenosine monophosphate deaminase 1

431

FABP3 - Fatty acid binding protein 3

432

SMTCK - sarcomeric mitochondrial creatine kinase

433

ENO3 – beta enolase

434

ACKNOWLEDGEMENTS

435

The authors extend our appreciation to the individuals who helped collect our samples: Lyn Hill,

436

Brenda Murdoch and Katherine Hunt. Finally, we would like to thank Washington Beef in


Page 21 of 36


437

Toppenish, WA for providing the carcasses for sampling, facilitating our tracking and sampling

438

of carcasses and providing the carcass grade data.

439

SUPPORTING INFORMATION DESCRIPTION

440

A table describing specific information about the primary and secondary antibodies can be found

441

in the supporting information.

442

FUNDING SOURCES

443

We gratefully acknowledge the financial support of this experiment by the Idaho Beef Council

444

through the Beef Checkoff program (IBC FY2010-BGK651) and the USDA Multi-State Hatch

445

Grant NC1184 through the University of Idaho Agricultural Experiment Station.



Page 22 of 36

446

REFERENCES

447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489

1. Bouley, J.; Chambon, C.; Picard, B., Mapping of bovine skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry. PROTEOMICS 2004, 4, 1811-1824. 2. Lawrie, R. A.; Ledward, D. A., Lawrie's Meat Science. 7 ed.; Woodhead Publishing Limited: Cambridge, 2006. 3. Jeremiah, L. E., A review of factors influencing consumption, selection and acceptability of meat purchases Journal of Consumer Studies & Home Economics 1982, 6, 137-154. 4. Boleman, S. J.; Boleman, S. L.; Miller, R. K.; Taylor, J. F.; Cross, H. R.; Wheeler, T. L.; Koohmaraie, M.; Shackelford, S. D.; Miller, M. F.; West, R. L.; Johnson, D. D.; Savell, J. W., Consumer evaluation of beef of known categories of tenderness. J. Anim. Sci. 1997, 75, 1521-4. 5. Hocquette, J. F.; Lehnert, S.; Barendse, W.; Cassar-Malek, I.; Picard, B., Recent advances in cattle functional genomics and their application to beef quality. animal 2007, 1, 159173. 6. Hollung, K.; Veiseth, E.; Jia, X.; Færgestad, E. M.; Hildrum, K. I., Application of proteomics to understand the molecular mechanisms behind meat quality. Meat Science 2007, 77, 97-104. 7. Bjarnadóttir, S. G.; Hollung, K.; Hoy, M.; Veiseth-Kent, E., Proteome changes in the insoluble protein fraction of bovine Longissimus dorsi muscle as a result of low-voltage electrical stimulation. Meat Science 2012, 89, 143-149. 8. Jia, X.; Ekman, M.; Grove, H.; Færgestad, E. M.; Aass, L.; Hildrum, K. I.; Hollung, K., Proteome changes in bovine longissimus thoracis muscle during the early postmortem storage period. J. Proteome Res. 2007, 6, 2720-2731. 9. Bouley, J.; Meunier, B.; Chambon, C.; De Smet, S.; Hocquette, J. F.; Picard, B., Proteomic analysis of bovine skeletal muscle hypertrophy. PROTEOMICS 2005, 5, 490-500. 10. Keady, S. M.; Kenny, D. A.; Ohlendieck, K.; Doyle, S.; Keane, M.; Waters, S. M., Proteomic profiling of bovine M. longissimus lumborum from Crossbred Aberdeen Angus and Belgian Blue sired steers varying in genetic merit for carcass weight. J. Anim. Sci. 2013, 91, 654665. 11. Shibata, M.; Matsumoto, K.; Oe, M.; Ohnishi-Kameyama, M.; Ojima, K.; Nakajima, I.; Muroya, S.; Chikuni, K., Differential expression of the skeletal muscle proteome in grazed cattle. J. Anim. Sci. 2009, 87, 2700-2708. 12. Jia, X.; Hildrum, K. I.; Westad, F.; Kummen, E.; Aass, L.; Hollung, K., Changes in Enzymes Associated with Energy Metabolism during the Early Post Mortem Period in Longissimus Thoracis Bovine Muscle Analyzed by Proteomics. J. Proteome Res. 2006, 5, 17631769. 13. Jia, X.; Hollung, K.; Therkildsen, M.; Hildrum, K. I.; Bendixen, E., Proteome analysis of early post-mortem changes in two bovine muscle types: M. longissimus dorsi and M. semitendinosis. PROTEOMICS 2006, 6, 936-944. 14. Lametsch, R.; Karlsson, A.; Rosenvold, K.; Andersen, H. J.; Roepstorff, P.; Bendixen, E., Postmortem Proteome Changes of Porcine Muscle Related to Tenderness. J. Agric. Food Chem. 2003, 51, 6992-6997. 15. Zhao, Y. M.; Basu, U.; Dodson, M. V.; Basarb, J. A.; Guan, L. L., Proteome differences associated with fat accumulation in bovine subcutaneous adipose tissues. Proteome Sci 2010, 8, 14.


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16. Mao, Y.; Hopkins, D. L.; Zhang, Y.; Li, P.; Zhu, L.; Dong, P.; Liang, R.; Dai, J.; Wang, X.; Luo, X., Beef quality with different intramuscular fat content and proteomic analysis using isobaric tag for relative and absolute quantitation of differentially expressed proteins. Meat Science 2016, 118, 96-102. 17. Bowker, B. C.; Eastridge, J. S.; Solomon, M. B., Measurement of muscle exudate protein composition as an indicator of beef tenderness. J. Food Sci. 2014, 79, C1292-C1297. 18. Joseph, P.; Suman, S. P.; Rentfrow, G.; Li, S.; Beach, C. M., Proteomics of musclespecific beef color stability. J. Agric. Food Chem. 2012, 60, 3196-3203. 19. Kim, N. K.; Cho, S.; Lee, S. H.; Park, H. R.; Lee, C. S.; Cho, Y. M.; Choy, Y. H.; Yoon, D.; Im, S. K.; Park, E. W., Proteins in longissimus muscle of Korean native cattle and their relationship to meat quality. Meat Science 2008, 80, 1068-1073. 20. Wei, J.; Kang, H. W.; Cohen, D. E., Thioesterase superfamily member 2 (Them2)/acylCoA thioesterase 13 (Acot13): a homotetrameric hotdog fold thioesterase with selectivity for long-chain fatty acyl-CoAs. Biochem. J. 2009, 421, 311-322. 21. Savitt, J. M.; Dawson, V. L.; Dawson, T. M., Diagnosis and treatment of Parkinson disease: molecules to medicine. The Journal of Clinical Investigation 2006, 116, 1744-1754. 22. Ozawa, S.; Mitsuhashi, T.; Mitsumoto, M.; Matsumoto, S.; Itoh, N.; Itagaki, K.; Kohno, Y.; Dohgo, T., The characteristics of muscle fiber types of longissimus thoracis muscle and their influences on the quantity and quality of meat from Japanese Black steers. Meat Science 2000, 54, 65-70. 23. Ryu, Y. C.; Kim, B. C., The relationship between muscle fiber characteristics, postmortem metabolic rate, and meat quality of pig longissimus dorsi muscle. Meat Science 2005, 71, 351-357. 24. Kim, N.-K.; Lim, D.; Lee, S.-H.; Cho, Y.-M.; Park, E.-W.; Lee, C.-S.; Shin, B.-S.; Kim, T.-H.; Yoon, D., Heat Shock Protein B1 and Its Regulator Genes Are Negatively Correlated with Intramuscular Fat Content in the Longissimus Thoracis Muscle of Hanwoo (Korean Cattle) Steers. J. Agric. Food Chem. 2011, 59, 5657-5664. 25. Guillemin, N.; Bonnet, M.; Jurie, C.; Picard, B., Functional analysis of beef tenderness. J. Proteomics 2011, 75, 352-365. 26. Picard, B.; Berri, C. c.; Lefaucheur, L.; Molette, C.; Sayd, T.; Terlouw, C., Skeletal muscle proteomics in livestock production. Briefings in Functional Genomics 2011, 9, 259-278.



522 523 524 525 526 527 528 529

A

Page 24 of 36

FIGURES

B


Page 25 of 36


530

Figure 1. Images from the 2D-DIGE gel analysis. Panel A represent the steers and panel B the

531

heifers. Pooled samples from high quality animals are Cy3 labeled and thus fluorescence is

532

green; pooled samples from low quality animals are Cy5 labeled and thus fluorescence is red. An

533

internal standard consisting of pooled protein from all animals in the study is also included,

534

fluorescence can be seen in yellow. The number next to each spot identifies that individual

535

protein, data regarding each protein spot can be seen in Table 2.

536



High Quality Carcass

|

Page 26 of 36

Low Quality Carcass

AMPD1

87 KDa High Quality Carcass

|

Low Quality Carcass 45 KDa

SMTCK

537 538


Page 27 of 36


539

Figure 2. Figure 2.Representative Western blot images from the heifers. The representative

540

images above represent an n = 5 for each group, high or low quality. High quality carcass

541

samples are on the left whereas low quality carcass samples are on the right. The two

542

representative proteins above are adenosine monophosphate deaminase 1 (AMPD1) and

543

sarcomeric mitochondrial creatine kinase (SMTCK). The actual molecular weight of each of the

544

proteins can be found to the right of the image.

545



High Quality Carcass

|

Page 28 of 36


CA3 High Quality Carcass

|


ENO3 High Quality Carcass

|

Low Quality Carcass

GPD1

36 KDa High Quality Carcass

|


PARK7

546 547


Page 29 of 36


548

Figure 3. Representative Western blot images from the steers. The representative images above

549

represent an n = 5 for each group, high or low carcass quality. High quality samples are on the

550

left whereas low quality carcass samples are on the right. The 4 representative proteins above are

551

carbonic anhydrase 3 (CA3), beta enolase (ENO3), glyceraldehyde 3 phosphate dehydrogenase

552

(GPD1), and parkinsons 7 (PARK7). The actual molecular weight of each of the proteins can be

553

found to the right of the image.

554



555 556

Page 30 of 36

TABLES Table 1. Carcass Data of Animals Selected for Analysis Steersb Low Quality

High Quality

pValued

High Quality

Heifersb Low Quality

pValued

HCW, kga

386.6 ± 13.6

353.6 ± 7.2

0.35

332.2 ± 11.8 309.7 ± 15.6

0.28

Yield Gradea

2.69 ± 0.22

2.27 ± 0.35

0.33

2.46 ± 0.23

2.10 ± 0.23

0.31

REA, cm2a

77.9 ± 3.7

84.3 ± 4.5

0.30

85.2 ± 2.7

82.3 ± 3.7

0.53

Marbling Scoreac

663.0 ± 12.2

697 ± 11.6

332.8 ± 7.2 < 0.0001

322.2 ± 4.6 < 0.0001

1 prime 4 prime 12 low 12 low select 11 choice 8 choice select a Values indicate average ± SEM; hot carcass weight (HCW); ribeye area (REA) b N=24 c Marbling score was provided by Washington Beef with marbling scores ranging from 300, indicating a slight degree of marbling and a quality grade of select, to 999, indicating an abundant degree of marbling and a quality grade of prime. d Difference between high and low quality carcasses, significant differences, p ≤ 0.05, are indicated in bold Quality Grade

557


Page 31 of 36


Table 2. 2D-DIGE Proteomics Results Spot Number

Protein Identification

Accession numbera

Heat Shock Proteins and Oxidative Protection heat shock protein beta-1 44 gi|71037405 heat shock protein beta-1 45 gi|71037405 protein DJ-1 52 gi|62751849 alpha-crystallin B chain 53 gi|27805849

Number Matched Peptidesc

Steers LQ/HQd

Heifers LQ/HQd

5.77 5.77 6.84 6.76

17 17 14 15

1.24 1.37 1.26 1.39

1.59 1.43 1.45 1.71

5.57

27

-1.27

-1.66

5.60

42

1.28

1.09

7.44

21

1.24

1.26

5.93

18

1.33

1.18

8.37

14

-1.26

-1.55

5.57

11

-1.44

-1.55

5.23

18

-1.29

1.18

5.31

19

-1.02

1.27

4.61 4.69

16 14

-1.04 -1.30

1.28 -1.11

4.64

15

-1.29

-1.13

5.71

11

1.13

1.36

6.42

10

1.20

1.74

7.74

12

-1.06

1.39

7.74

12

1.03

1.26

5.71

10

-1.09

1.46

4.96

13

2.39

2.10

4.96

14

1.55

1.44

4.96

13

-1.28

-1.05

5.40

15

-1.58

1.00

4.86

12

1.43

1.58

4.86

9

1.43

1.90

4.86

11

1.50

1.49

Protein Protein MW, Dab pIb 22665.4 22665.4 20022.6 20024.4

Cytoskeletal Proteins: Muscle maturity and fiber type 1 myosin-1 gi|41386691 222851.5 myosin-binding protein 2 gi|160425243 133896.5 C, slow-type myosin-binding protein 3 gi|133908641 127991.4 C, fast-type myosin-binding protein 4 gi|311258038 76122.0 C, fast-type TRIO and F-actin gi|114686389 240783.2 5 binding protein 7 myosin-1 gi|41386691 222851.5 Actin, alpha 1, skeletal 26 gi|134024776 41995.9 muscle actin, alpha skeletal 27 gi|27819614 42108.9 muscle 29 tropomyosin beta chain gi|73971292 31546.9 30 tropomyosin alpha chain gi|63252898 32688.7 skeletal muscle 31 gi|339956 30362.4 tropomyosin troponin T, slow skeletal 32 gi|41386697 31265.1 muscle troponin T fast skeletal 33 gi|21038996 31490.3 muscle type troponin T, fast skeletal 34 gi|47824864 29800.5 muscle troponin T, fast skeletal 36 gi|47824864 29800.5 muscle troponin T, slow skeletal 38 gi|41386697 31265.1 muscle myosin light chain 1/3, 42 gi|118601750 20918.6 skeletal muscle myosin light chain 1/3, 43 gi|118601750 20918.6 skeletal muscle myosin light chain 1/3, 50 gi|118601750 20918.6 skeletal muscle 51 myosin light chain 6B gi|115496556 23388.2 myosin regulatory light 56 gi|296478466 18892.4 chain 2, cardiac myosin regulatory light 57 gi|296478466 18892.4 chain 2, cardiac myosin regulatory light 58 gi|296478466 18892.4 chain 2, cardiac



62 63 69

myosin regulatory light chain 2, skeletal myosin regulatory light chain 2, skeletal myosin light chain 1, skeletal muscle

Skeletal muscle energetics AMP deaminase 1 9 pyruvate kinase, muscle 16 pyruvate kinase, muscle 18 Sarcomeric mitochondrial 24 creatine kinase adenylate kinase 55 isoenzyme 1 Aconitase 2, 8 mitochondrial succinate dehydrogenase, 12 subunit A phosphoglucomutase-1 13 phosphoglucomutase-1 14 phosphoglucomutase-1 15 glucose-6-phosphate 17 isomerase aldehyde dehydrogenase 19 1 alpha-enolase 20 beta-enolase 21 beta-enolase 22 beta-enolase 23 phosphoglycerate kinase 25 1 fructose-bisphosphate 28 aldolase C-A glycerol-3-phosphate 35 dehydrogenase glyceraldehyde-337 phosphate dehydrogenase C->U-editing enzyme 39 APOBEC-2 carbonic anhydrase 3 40 carbonic anhydrase 3 41 triosephosphate 46 isomerase triosephosphate 47 isomerase triosephosphate 48 isomerase triosephosphate 49 isomerase Miscellaneous Proteins 6 albumin protein 10 serotransferrin precursor

Page 32 of 36

gi|115497166

19000.4

4.91

20

-1.53

-1.52

gi|115497166

19000.4

4.91

18

-1.40

-1.57

gi|115304798

19492.6

4.73

6

-1.30

-1.53

gi|154152079 gi|194670470 gi|194670470

86575.1 57912.0 57912.0

6.92 7.96 7.96

13 26 26

-1.24 1.26 1.37

-1.61 1.25 1.38

gi|60097925

42961.8

6.63

21

1.34

1.41

gi|61888850

21650.3

8.40

18

1.29

1.26

gi|74268076

85233.9

8.08

34

-1.00

1.33

gi|152941202

72886.2

7.55

31

-1.03

1.33

gi|116004023 gi|116004023 gi|116004023

61550.8 61550.8 61550.8

6.36 6.36 6.36

31 31 30

-1.44 1.00 1.66

1.08 1.43 1.03

gi|296477774

62815.0

7.33

24

1.23

1.26

gi|2183299

54799.9

6.30

12

-1.25

-1.34

gi|296479148 gi|77736349 gi|77736349 gi|77736349

47254.3 47066.4 47066.4 47066.4

6.37 7.60 7.60 7.60

23 20 24 24

1.17 1.29 1.38 1.39

1.31 1.27 1.37 1.37

gi|77735551

44509.1

8.48

16

1.22

1.27

gi|156120479

39411.3

8.45

20

1.17

1.30

gi|78365297

37665.4

6.42

15

1.26

1.30

gi|65987

35686.2

6.90

14

1.21

1.29

gi|77735661

25945.9

4.84

15

1.19

1.28

gi|77735829 gi|77735829

29351.8 29351.8

7.71 7.71

18 20

-1.37 -1.10

-1.29 -1.41

gi|61888856

26672.8

6.45

23

1.33

1.24

gi|61888856

26672.8

6.45

23

1.34

1.31

gi|61888856

26672.8

6.45

21

1.27

1.18

gi|61888856

26672.8

6.45

22

1.31

1.13

gi|74267962 gi|114326282

69190.4 77688.7

5.88 6.75

9 37

2.10 -1.31

-1.43 1.10


Page 33 of 36


11

serotransferrin precursor gi|114326282 77688.7 6.75 32 -1.26 1.37 phosphatidylethanolamine 54 gi|157829678 20713.5 6.62 15 1.50 1.73 binding protein 59 myoglobin gi|27806939 17066.9 6.90 15 -1.07 1.46 60 myoglobin gi|27806939 17066.9 6.90 15 1.24 1.64 61 myoglobin gi|27806939 17066.9 6.90 14 1.44 1.18 64 myoglobin gi|27806939 17066.9 6.90 13 -1.26 -1.13 65 myoglobin gi|27806939 17066.9 6.90 14 -1.47 -1.41 66 myoglobin gi|27806939 17066.9 6.90 14 1.18 -1.25 67 myoglobin gi|27806939 17066.9 6.90 16 -1.33 -1.48 68 myoglobin gi|27806939 17066.9 6.90 15 -1.34 -1.21 fatty acid-binding 70 gi|27805809 14769.6 6.73 14 -1.28 1.07 protein, heart 71 hemoglobin beta gi|294459577 15969.2 6.36 10 -1.61 -3.65 72 hemoglobin, beta subunit gi|27819608 15944.3 7.01 16 -2.25 -1.63 73 hemoglobin, beta subunit gi|27819608 15944.3 7.01 16 -2.12 -1.34 74 hemoglobin, beta subunit gi|27819608 15944.3 7.01 17 1.01 1.28 hemoglobin, alpha 75 gi|116812902 15174.9 8.07 11 -1.64 -1.27 subunit 76 hemoglobin, beta subunit gi|27819608 15944.3 7.01 15 -1.10 -1.40 77 hemoglobin, beta subunit gi|27819608 15944.3 7.01 16 -2.34 -1.79 a b Protein ID and Accession numbers were derived from NCBI databases The theoretical molecular weight (MW) and isoelectric point (PI) of the identified spots were derived from NCBI databases cNumber of matched peptides dIndicates fold change difference, positive values represent greater abundance in the longissimus thoracis of high carcass quality grade (HQ) animals and negative values represent greater abundance in the longissimus dorsi of low carcass quality grade animals. All protein spots shown in the table had a MASCOT protein score C.I. % of 99.7 or higher.

558



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Table 3. Western Blot Analyses of Longissimus thoracis in the Heifers N = 24 High Qualitya

Protein AMPD1c CA3d

165.1 ± 17.2

N = 48

Low Qualitya p Valueb

High Qualitya

Low Qualitya p Valueb

134.4 ± 10.4

114.9 ± 9.5 148.7 ± 3.1

0.03 0.22

171.6 ± 10.3 146.2 ± 6.6

128.9 ± 8.8 153.8 ± 2.7

0.005 0.30

SMTCK ENO3f

100.5 ± 18.8 181.3 ± 8.6

157.9 ± 16.3 193.2 ± 7.0

0.04 0.30

121.3 ± 11.5 163.9 ± 12.5 178.0 ± 4.9 189.6 ± 6.4

0.01 0.16

FABP3g GPD1h

156.6 ± 2.3 108.8 ± 9.7

164.5 ± 8.4 112.4 ± 7.3

0.39 0.77

138.2 ± 6.9 109.0 ± 5.5

157.0 ± 9.5 113.3 ± 6.0

0.12 0.61

HSPB1i MYH1j PARK7k TPIl TPMm

197.7 ± 5.0 160.8 ± 6.5 115.8 ± 13.5 174.8 ± 9.5 196.6 ± 7.3

189.8 ± 10.9 157.3 ± 10.4 96.9 ± 4.2 185.8 ± 16.3 206.9 ± 12.6

0.52 0.78 0.23 0.57 0.50

209.5 ± 5.4 208 ± 7.7 151.6 ± 4.5 151.8 ± 5.4 122.8 ± 8.1 109.5 ± 5.13 145.2 ± 10.7 171.1 ± 12.9 181.9 ± 6.1 202.8 ± 8.1

0.87 0.98 0.18 0.14

e

a

2

0.05

b

Indicates relative densitometric values (ODU/mm ) obtained from the Western blot ± SEM. p ≤ 0.05 was considered significant and is indicated in bold. c adenosine monophosphate deaminase d carbonic anhydrase 3 e sarcomeric mitochondrial creatine kinase f beta enolase g fatty acid binding protein 3 h glyceraldehyde 3 phosphate dehydrogenase i heat shock protein beta 1 j myosin heavy chain 1 k parkinsons 7 l triose phosphate isomerase m tropomyosin

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Table 4. Western Blot Analyses of the Longissimus thoracis in the Steers

Protein AMPD1c CA3

d

SMTCKe ENO3

f

FABP3

g

GPD1h

N = 24 N = 48 a b a High Quality Low Quality p Value High Quality Low Qualitya p Valueb a

188.8 ± 13.0

156.7 ± 7.4

0.05

175.4 ± 8.9

154.5 ± 9.1

0.12

126.8 ± 6.1

149.3 ± 6.0

0.02

122.3 ± 4.5

151.7 ± 4.7

0.0002

176.9 ± 25.6

194.5 ± 14.3

0.56

171.3 ± 13.6

199.0 ± 8.4

0.10

168.6 ± 9.2

211.2 ± 8.0

0.006

168.5 ± 5.8

215.9 ± 4.6

Analysis of Longissimus thoracis Protein Expression Associated with

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