Proteome Changes in Bovine Longissimus Thoracis Muscle During

Proteome Changes in Bovine Longissimus Thoracis Muscle During the Early Postmortem Storage Period. Xiaohong ... Publication Date (Web): June 14, 2007...
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Proteome Changes in Bovine Longissimus Thoracis Muscle During the Early Postmortem Storage Period Xiaohong Jia,†,‡ Maria Ekman,† Harald Grove,†,‡ Ellen M. Færgestad,† Laila Aass,§ Kjell I. Hildrum,† and Kristin Hollung*,† Matforsk AS, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway, and Department of Chemistry, Biotechnology and Food Science, and Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway Received March 27, 2007

Postmortem changes in protein composition up to 24 h in bovine longissimus thoracis muscle were investigated by two-dimensional gel electrophoresis and MALDI-TOF MS/MS. A total of 47 spots were significantly changed the first 24 h postmortem. The 39 identified proteins can be divided into five groups: metabolic enzymes, defense and stress proteins, structural proteins, proteolytic enzymes, and unclassified proteins. The identified metabolic enzymes are all associated with ATP-generating pathways, either the glycolytic pathway or energy metabolism. In addition, several defense and stress proteins were changed in abundance in this study. These findings contribute to a better understanding of the biochemical processes during postmortem storage of meat. Keywords: Bovine muscle • proteomics • meat quality • postmortem

Introduction Variability of meat quality is a major concern for both the meat industry and the consumer. Many factors can affect final meat quality, such as feeding and transport before slaughter, slaughter conditions, electrical stimulation, and chilling conditions. In recent years proteomics has been applied to study protein expression in muscle, in order to better understand the biochemical processes taking place during postmortem storage of meat. Previous proteome analyses of pork1-4 and beef5,6 muscles have shown that many metabolic proteins change in abundance during postmortem storage. The metabolic proteins and proteases are especially interesting, since they are crucial in controlling biochemical and biophysical processes in muscle cells and in degradation of myofibril proteins during the meat tenderization processes. A comparison of metabolic protein composition in bovine longissimus thoracis muscle biopsies taken from live animals and samples collected shortly after slaughter revealed that a wide range of metabolic enzymes and stress proteins increased in abundance after slaughter.6 Several of these proteins were glycolytic enzymes such as enolase, aldehyde dehydrogenase, phosphoglycerate kinase, and enzymes involved in oxidative metabolism, such as ATP-specific succinyl-CoA synthetase and isocitrate dehydrogenase.6 These findings support an expected shift in energy metabolism in * To whom correspondence should be addressed. Fax: +47 64970333. Tel: +47 64970142. E-mail: [email protected]. † Matforsk AS. ‡ Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences. § Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences.

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postmortem muscle toward the glycolytic pathway and also an increase in aerobic energy metabolism the first hour after slaughter. In another study the changes in bovine longissimus thoracis muscle proteome between slaughter and 24 h storage was investigated, and changes were observed in both metabolic enzymes and stress proteins.5 To obtain more information on the metabolic changes occurring during the early postmortem period in bovine longissimus thoracis muscle, samples collected at 1, 2, 3, 6, 10, and 24 h after slaughter were analyzed.

Materials and Methods Animals and Sampling. The experiment included eight NRF (Norwegian Red) dual-purpose young bulls (approximately 13 months of age/450 kg live weight) from a performance test station (GENO-Breeding and AI Association) in 2003. The bulls were transported (1 h) to a commercial slaughterhouse (Gilde HedOpp, Norway) and slaughtered shortly after arrival. The carcasses were electrically stimulated (90 V) approximately 30 min postmortem. The hot boned M. longissimus thoracis (LT) were packed and kept at 12 °C for the first 10 h and at 4 °C for the rest of the storage period. A piece of muscle tissue was taken at 1, 2, 3, 6, 10, and 24 h postmortem, snap frozen in liquid nitrogen, and stored at -80 °C for further analysis. The experiment included six sampling times after slaughter (1, 2, 3, 6, 10, and 24 h) on eight animals (biological replicates). Each sample was extracted once, while the subsequent 2-DE analysis of the samples were conducted twice in subsequent order giving a total of 96 gels [6 sampling times × 8 biological replicates (animals) × 2 technical replicates]. Each gel batch consisted of replicates of all time points from one animal. 10.1021/pr070173o CCC: $37.00

 2007 American Chemical Society

Proteome Changes in Bovine Longissimus Thoracis

Extraction of Proteins. Pieces (200 mg) of frozen muscle tissue were homogenized in 1 mL of TES buffer (10 mM Tris, pH 7.6, 1 mM EDTA, and 0.25 M sucrose) by using an Ultraturrax (Polytron PT, Kinematica AG, Luzern, Switzerland) at 12 000 rpm for 3 × 20 s. The homogenate was transferred to Eppendorf tubes and centrifuged (30 min at 10 000 rpm) at 4 °C to remove TES-insoluble proteins. Protein concentrations were measured with a commercial kit at 760 nm (RC DC Protein Assay, Bio-Rad) in a spectrophotometer (Ultrospec 3000, Pharmacia Biotech) using BSA as standard. Two-Dimensional Gel Electrophoresis. 2-DE was performed according to the method described previously.1 Immobilized pH gradient (IPG) strips (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) spanning the pH region 4-7 including many of the metabolic enzymes were used in the first dimension. For analytical 2-DE, 70 µg of proteins was loaded on each IPG strip by in-gel rehydration, while 500 µg of proteins was loaded for preparative 2-DE. The isoelectric focusing was performed on the Pharmacia Multiphor unit equipped with a Heto temperature controller. Low voltage (50 V) was applied in the initial step followed by a stepwise increase to 3500 V, reaching a total of 70 000 V h. Proteins were separated on 12.5% SDSPAGE in the second dimension using the Ettan Dalttwelve large format vertical system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The analytical gels were stained by silver staining as described by Blum,7 while the preparative gels were stained according to Shevchenko.8 Several preparative gels were made for repeated identification of the proteins. Image Analysis and Data Analysis. The 2-DE gels were scanned using an office scanner (Epson Expression 1680 Pro, Epson) and analyzed using the ImageMaster 2D Platinum software Version 6.0 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). A reference gel was selected to match all the other gels. Manual editing was performed to remove artifacts and mismatched spots. The protein spots were matched across all 96 gels and the resulting table was imported into Unscrambler version 9.2 (CAMO A/S), Matlab version 7.0.4 (The Mathworks Inc.), and 50-50 MANOVA (http://www.matforsk.no/ola/ffmanova.htm) for statistical analysis. Analysis of spot volumes from 2-DE experiments is challenging,9 due to potential erroneous missing values. As we in this experiment expect a number of truly absent proteins at some time points, we have chosen to first insert the value zero for missing values in the statistical analysis. Thereafter, all proteins found to be significantly related to the time after slaughter were tested by leaving the missing values as missing. Technical reproducibility and resolution of the 2-D gels were satisfactory for all gels. However, the technical replicate 1 was more homogeneous than the technical replicate 2. We therefore chose to perform statistical analysis within replicate 1. Thereafter those proteins found to be significant in replicate 1 were also tested in replicate 2. We only report those proteins that could be verified in both replicates. The sampling time 24 h was clearly distinct from the other time points. This sampling date was therefore excluded from statistical analysis, but included in the time course plot of significant proteins. The multivariate data analysis PCA was conducted to visualize the main variations in the proteome and to detect clusters and structures in the data.10 The multivariate regression approach, partial least-squares regression (PLSR)10 was performed to relate the protein variables, as regressor variables (xvariables), to the sampling times (y-variables). Both PCA and PLSR transform the original data down to a smaller set of

research articles variables called principal components (PCs), which are linear combinations of the original variables containing the relevant information in the original data. By PCA the PCs are estimated to account for, in decreasing order, the largest variation in the original variables within one block of data, whereas PLSR is a supervised method where the PCs in decreasing order are the most relevant for predicting the y-variables from the x-variables. The PCs can be viewed in a 2-D plot where the samples are plotted according to the new coordinates from the transformed data set and a corresponding plot of variables. This gives an overview of the main structure of the data, and the relation between protein compositions. The model was evaluated by cross validation keeping out one animal at the time.10 To identify proteins changing significantly with time, we first conducted a univariate F-test (using 50-50 MANOVA) with sampling time, time × time, animal, and the interaction between time and animal as regressors and protein volume as response, and those proteins classified as significant at the 5% level were kept for further analysis. Variables found to be significant by the univariate test was tested by PLSR using a cross-model validation (CMV) procedure, which uses a combination of PLSR and Jack-Knife adapted to bilinear methods and t-tests of the regression coefficients to validate the stability of the regression coefficient over different subsets of samples. The CMV is a two-layer cross-validation method, where one or more samples are left out while the rest of the samples are subject to cross-validation. The criterion for selecting variables is to select only the variables that are found significant in a certain number of all individual cross-validations inside the CMV. The cross-validation procedure was performed by keeping out all samples from one animal while calculating a model based on the other animals. The list of proteins considered significantly changed between the sampling times was based on those proteins being significant in at least 70% of the crossvalidation steps in the CMV. Proteins at 70% significance level from the technical replicate 1 were validated in the technical replicate 2 by ANOVA. The final list includes proteins that were significantly changed (p < 0.05) in both technical replicates. Protein Identification. The protein spots of interest were cut out of the preparative gels using pipet tips and extracted from gels according to the method of Jensen.11 Briefly, the washing/dehydration process was carried out by adding 150 µL of 50% v/v ACN and shaking 15 min at room temperature before the gels were dried in a SpeedVac centrifuge (ISS 110 SpeedVac System, Thermo Savant) for 30 min. Then 150 µL of 10 mM DTT was added, and gels were incubated for 45 min at 56 °C, followed by addition of 150 µL of 55 mM iodoacetamide and incubated for 30 min at room temperature in the dark. Afterward the plugs were washed with 50% v/v ACN and dried. Trypsin digestion buffer (30 µL, 5 ng/µL) was added to the dried gel pieces and incubated on ice for 30 min and at 37 °C overnight. A column consisting of 100-300 nL of Poros reverse phase R2 (20-30 µm bead size, PerSeptive Biosystem, Wellesley, MA) was packed in a constricted GeLoader tip (Eppendorf, Hamburg, Germany). A 10 mL syringe was used to force liquid through the column. Then 20 µL of the tryptic protein digests was loaded onto the column and washed with 20 µL of 0.1% TFA. The peptides were eluted with 0.8 µL of matrix solution [5 mg/mL R-cyano-4-hydroxy-trans-cinnamic acid (Agilent Technoligies, Inc.) in 70% ACN/0.1% TFA] and spotted directly on the MALDI plate. Journal of Proteome Research • Vol. 6, No. 7, 2007 2721

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Figure 1. Silver-stained 2-DE gel images of bovine longissimus thoracis at different time points postmortem: (A) 1 h, (B) 2 h, (C) 3 h, (D) 6 h, (E) 10 h, and (F) 24 h. Protein (70 µg) was loaded, and 2-DE was performed using a pH range of 4-7 in the first dimension and SDS-PAGE (12.5% T) in the second dimension.

An Ultraflex MALDI-TOF/TOF mass spectrometer with the LIFT module (Bruker Daltonics) was used for protein identification. A peptide mixture (peptide calibration standard I, Bruker Daltonics) was used for external calibration, while the internal calibration was performed using the trypsin autolysis products. The program package FlexAnalysis 2.4 (Version 1.1.3, Bruker Daltonics) was used to create the peak list using median baseline subtraction with 0.8 in flatness and smoothing by the Savitzky-Golay filter of 0.2 m/z in width. BioTools 3.0 (Version 1.0, Bruker Daltonics) was used for interpretation of MS and MS/MS spectra. Proteins were identified by peptide mass fingerprinting (PMF) using the database search program MASCOT (http://www.matrixscience.com/). An accuracy of 100 ppm (parts per million) was used in the search criteria. Fixed modification and variable modification used were carbamidomethyl (C) and oxidation (M), respectively. MS/MS analysis and repeated Mascot-based database searches of a minimum of three precursor ions recognized in the PMF search were performed to confirm the PMF-based protein identification. The number of peptide matches, sequence coverage, molecular weight, and pI were used to evaluate the database search results.

Results Postmortem changes up to 24 h in bovine muscles were analyzed by 2-DE-based proteomics. Figure 1 shows represen2722

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tative 2-DE gels corresponding to samples collected at 1, 2, 3, 6, 10, and 24 h postmortem from one animal. The proteins in the molecular mass region from 10 to 75 kDa and the pH range between 4 and 7 were included in the comparative analyses. Most of the soluble proteins were observed between pH 5 and 7. Image analysis allowed matching of 974 spots across all 96 gels. By PCA performed within each replicate, the samples were separated according to time postmortem along the first and most important PC (Figure 2A). Thus, despite the large variation typically found in the 2-DE pattern, the time after slaughter was manifested as the largest variation, as this was reflected by the first PC. For replicate 1 shown in Figure 2A, PC1 and PC2 explain 12% and 9% of the total variance in the proteome data, respectively, and by 10 PCs 57% of the variation is captured. A number of proteins contributed to this variation, as seen in the corresponding loading plot of the spot volumes (Figure 2B). The moderate degree of explained variance of PC1 and PC2 is what usually can be expected for 2-DE data, due to the high noise level in the images.12 When the data were modeled by PLSR using the sampling time as response (y-variables) and the protein intensities as regressor variables (x-variables), the first four components in the PLSR model explained 73% of the total explained validated variation. The pattern of the samples was similar in the PLS score plot (Figure 2C), for PCA (Figure 2A), reflecting variation

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Figure 2. Score plot and loading plot of PC1 and PC2 from principal component analysis (PCA) (A and B) and partial least-squares regression (PLSR) (C and D) of 974 matched spots (replicate 1) from 2-DE analysis of bovine longissimus thoracis muscle collected at 1, 2, 3, 6, and 10 h postmortem. Explained variance for each PC is given on the axes, where validated variance is given for PLSR. 2724

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Figure 3. Silver-stained 2-DE gel of bovine longissimus thoracis. Protein (70 µg) was loaded, and 2-DE was performed using a pH range of 4-7 in the first dimension and SDS-PAGE (12.5% T) in the second dimension. Protein spots that are changed according to the postmortem sampling times are numbered.

in the time postmortem in the first component. The loading plot of the PLSR model (Figure 2D) reflects variation in a large number of proteins along this component. Among the 974 protein variables from the first replicate, 327 were found to be significantly changed at the 5% level using univariate ANOVA. When analyzing these proteins further by CMV 154 variables was significant in at least 70% of the crossvalidation steps. Of the 154 variables from the technical replicate 1, 105 proteins could be matched to proteins in the replicate 2 data set. ANOVA analysis of these 105 proteins, gave 47 found to change (p < 0.05) in both replicates, and these were selected for identification. Figure 3 shows a representative gel where these 47 significantly changed spots are numbered. A total of 39 spots were successfully identified by MALDITOF MS/MS (Table 1). Three protein spots could not be detected in the preparative gels and were therefore not identified, and five spots were not identified due to low protein concentration. Among the identified 39 spots, 33 were identified by matching peptide data to bovine protein sequences in the data base, while seven spots were identified due to interspecies homology to human and mouse protein sequences (Table 1).

Spot 1140 harbors a mixture of two proteins: adenylate kinase 1 and creatine kinase M chain (fragment). For some protein spots a moderate shift in molecular weight and pI value (spots 210, 342, 413, 418, 438, 511, 626, 647, 656, 896, 1006, 1020, 1087, 1093, 1140, 1141, 1163, 1213, 1294, and 1275) compared to the theoretical pI and MW were observed. Several sets of multiple spots harboring the same protein were identified, namely, tubulin (spots 294, 295 and 299), ADP-ribosylhydrolase like 1 (spots 647 and 656), GPD1 protein (spots 770, 780 and 788), HSPB1 protein (spots 1077, 1078, 1080, 1085 and 1086), and DJ-1 protein (spots 1154, 1158, 1161 and 1167). The theoretical MW and pI of tubulin, HSPB1 protein, and DJ-1 protein all differed from the experimental MW and pI (Table 1). The identified proteins can be divided into five groups: metabolism, defense and stress, cell structure, protease, and other unclassified proteins. Proteins are classified according to proposed functions based on Swiss-Prot, NCBI and TrEMBL protein databases. The expression profiles of the 39 identified proteins are shown in Figure 4. Of the metabolic proteins (Figure 4A), spot 342, 896, and 1140 increased in intensities up to 24 h, while spot 210 decreased down to 24 h. The profiles Journal of Proteome Research • Vol. 6, No. 7, 2007 2725

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Table 1. Proteins in Bovine Longissimus Thoracis Changed in Abundance During Postmortem Storagea spots

210 342 418 647 656 770 780 788 896 1006 1140 1140 1077 1078 1080 1085 1086 1119 1221 1154 1158 1161 1167 1020 1141 1163 1093 1275 294 295 299 511 1233 1213

413 438 626 1087 1166 1294 a

identified proteins

experimental pI/Mr

matched peptides/ % sequence coverage

Metabolic Enzymes 5.70/60 000 15/33 6.60/55 000 21/43 6.40/50 000 13/33 5.60/40 000 10/23 5.70/41 000 15/29 6.70/35 000 22/69 6.65/35 000 21/70 6.60/35 000 18/55 5.80/31 000 11/38 5.70/27 000 11/49 6.50/23 000 16/62 6.50/23 000 15/28 Cellular Defense/Stress Proteins HSPB1 protein 5.60/26 000 10/65 HSPB1 protein 5.40/26 000 6/36 HSPB1 protein 5.70/26000 9/45 HSPB1 protein 5.90/26 000 11/73 HSPB1 protein 6.10/26 000 13/73 HSPB1 protein 6.55/24 000 9/47 MGC140541 protein 6.30/19 000 10/64 PARK7 protein (DJ-1) 6.30/22 500 11/56 PARK7 protein (DJ-1) 5.90/22 500 19/75 PARK7 protein (DJ-1) 6.60/22 500 20/82 PARK7 protein (DJ-1) 6.40/22 500 14/59 antioxidant protein 2 6.10/27 000 10/45 substrate protein of mitochondrial ATP-dependent 4.20/24 000 6/56 proteinase SP-22 peroxiredoxin 2 5.60/22 500 7/39 biliverdin reductase B (flavin reductase (NADPH)) 6.70/24 000 14/77 ubiquitin-conjugating enzyme E2N 5.60/17 000 12/61 Cell Structure Proteins tubulin, R4 5.10/60 000 18/46 tubulin, R8 5.20/60 000 9/30 tubulin, R8 5.30/60 000 14/34 actin, R1, skeletal muscle 5.25/42 500 24/64 cofilin 2 5.90/17 000 10/55 Proteolytic Enzyme proteasome subunit, β type, 2 miscellaneous 6.75/21 000 9/51 proteins Miscellaneous Proteins chain A, cytochrome bc1 complex 5.50/50 000 12/35 eukaryotic translation initiation factor 4AII 5.30/48 000 17/37 hypothetical protein LOC537301 5.25/42 000 22/53 growth factor receptor-bound protein 2 isoform 1 6.30/26 000 10/45 tumor protein, translationally controlled 1 4.80/23 000 9/41 hypothetical protein LOC533224 6.75/13 500 9/67 chain d, aldehyde dehydrogenase chain d, aldehyde dehydrogenase enolase 1 ADP-ribosylhydrolase like 1 ADP-ribosylhydrolase like 1 GPD1 protein GPD1 protein GPD1 protein 3-hydroxyisobutyrate dehydrogenase guanidinoacetate N-methyltransferase adenylate kinase 1, soluble creatine kinase M chain

NCBI accession no. (source)

theoretical pI/Mr

gi|2624889 (bovine) gi|2624889 (bovine) gi|87196501 (bovine) gi|73586801 (bovine) gi|77736015 (bovine) gi|88682930 (bovine) gi|88682930 (bovine) gi|88682930 (bovine) gi|88682977 (bovine) gi|83638578 (bovine) gi|92097535 (bovine) gi|4838363 (bovine)

6.05/54 859 6.05/54 859 6.37/47 639 5.60/40 368 5.60/40 368 6.14/37 549 6.14/37 549 6.14/37 549 8.38/35 786 5.70/26 821 8.40/21 764 6.63/43 172

gi|74354863 (bovine) gi|74354863 (bovine) gi|85542053 (bovine) gi|85542053 (bovine) gi|74354863 (bovine) gi|85542053 (bovine) gi|109939807 (bovine) gi|73586598 (bovine) gi|73586598 (bovine) gi|73586598 (bovine) gi|73586598 (bovine) gi|27807167 (bovine) gi|627764 (bovine)

5.98/22436 5.98/22 436 5.98/22 436 5.98/22 436 5.98/22 436 5.98/22 436 5.95/17 515 6.84/19 769 6.84/19 769 6.84/19 769 6.84/19 769 6.00/25 108 5.73/21 709

gi|27807469 (bovine) gi|74354774 (bovine) gi|4507793 (human)

5.37/22 217 6.58/22 232 6.13/17 184

gi|6678467 (mouse) gi|8394493 (mouse) gi|8394493 (mouse) gi|27819614 (bovine) gi|33946278 (human)

4.95/50 634 4.97/50 704 4.97/50 704 5.31/42 451 7.66/18 839

gi|62751339 (bovine)

6.51/22 996

gi|4139392 (bovine) gi|485388 (human) gi|82697389 (bovine) gi|4504111 (human) gi|62177164 (bovine) gi|82697371 (bovine)

5.46/49 866 5.33/46 593 5.30/42 991 5.89/25 304 4.84/19 683 6.73/13 494

Of the 47 spots changed during postmortem storage of beef with 5% significantly level, we identified 39 spots. Eight spots could not be identified.

of spots 418, 770, 788, and 1006 decreased after 6 h postmortem. For the defense and stress proteins, spots 1077, 1078, 1080, 1085, and 1086 decreased, while the rest of the spots increased in abundance postmortem (Figure 4B). The structural proteins decreased in abundance postmortem (Figure 4C), while the proteasome subunit decreased the first hours and then increased at 24 h (Figure 4D). The unclassified protein group (Figure 4E) contains spots that decreased (spots 413, 438, and 1087) in abundance or increased (spots 1166 and 1294) up to 10 h postmortem.

Discussion We have investigated changes in postmortem bovine muscle and identified and characterized changes during postmortem storage. In the present study, we observed significant changes in 47 proteins, and among these we identified 39 proteins. Most of the abundance profiles were either gradually increasing or decreasing, giving a smooth curve. These proteins were found 2726

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to be significant in both replicates tested, using univariate ANOVA tests of the spot volumes as well by using multivariate test based on the stability of the regression coefficients. Postmortem Energy Metabolism. The glycolytic pathway plays a key role in energy metabolism by conversion of glucose to pyruvate while generating ATPs. Under aerobic conditions, pyruvate is oxidized to H2O and CO2 via the tricarboxylic acid cycle (TAC) and oxidative phosphorylation. Under anaerobic conditions, pyruvate is instead converted to lactate in muscle. Once an animal is slaughtered the blood and oxygen supply to the muscle stops. In the muscle some oxygen is stored in myoglobin,13 but gradually energy metabolism is switched from aerobic metabolism to anaerobic metabolism. The pH decline is a result of lactate production and accumulation within the muscles. The lactate production itself is caused by a forced requirement for ATP to sustain the biological processes in the cell. The two most important sources of ATP generation is degradation of glycogen to lactic acid with the release of

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Figure 4 (continued)

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Figure 4. Expression profiles of the identified proteins: (A) metabolic proteins, (B) cellular defense and stress proteins, (C) structure proteins, (D) proteasome, and (E) miscellaneous proteins. The x-axis corresponds to postmortem sampling times 1, 2, 3, 6, 10, and 24 h. The y-axis corresponds to the relative volume (%Vol) for each spot, which was calculated by dividing each spot volume by the total volume of all the spots in the 2-DE gel. 2728

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hydrogen ions, which will accumulate locally in the muscles and via transfer of phosphate from creatine phosphate to ADP to yield creatine and ATP.14 In this study, seven enzymes that are involved in energy metabolism significantly increased in abundance, namely, adenylate kinase 1, 3-hydroxyisobutyrate dehydrogenase, guanidonoacetate N-methyltransferase, creatine kinase M chain, glycerol-3-phosphate dehydrogenase 1, enolase 1, and aldehyde dehydrogenase. Enolase 1, aldehyde dehydrogenase, and 3-hydroxyisobutyrate dehydrogenase all increased in abundance during very early postmortem storage of beef and are involved in the enzymatic reactions of the glycolysis and TCA cycle. The glycolytic enzyme enolase 1 catalyzes the dehydration of 2-Pglycerate to P-enolpyruvate. NAD-dependent aldehyde dehydrogenase catalyzes the reaction between glyceraldehyde and glycerate, while 3-hydroxyisobutyrate dehydrogenase is involved in the degradation of valine to succinyl-CoA. Both glyceraldehyde and succinyl-CoA are further utilized in the glycolysis and TCA pathway. The intensity of enolase 1 increased to 4 h postmortem and then decreased during storage, indicating degradation after 4 h. Our previous proteome study in bovine muscle6 showed that enolase 1, 3-hydroxyisobutyrate dehydrogenase, and aldehyde dehydrogenase also increased in abundance immediately after slaughter. Two recent proteome studies in pigs indicate that enolase 1 and aldehyde dehydrogenase may be related to meat color development15 and to be affected by compensatory growth.16 Another source of ATP generation in muscle is via transfer of phosphate from creatine phosphate (CP) and ADP to yield creatine and ATP. Two enzymes associated with this process changed in abundance during postmortem storage. Guanidinoacetate N-methyltransferase catalyzes the last step of creatine biosynthesis and decreases in intensity, while creatine kinase reversibly catalyzes the transfer of phosphate between ATP and creatine phosphate and was identified as a fragment with increased intensity. Creatine kinase was degraded postmortem, and this could be caused by depletion of ATP. Creatine kinase has been reported to change in several proteome analyses in pigs related to postmortem storage,3 preslaughter conditions,4 and meat color.15 Three enzymes, namely, glycerol-3-phosphate dehydrogenase 1 (GPD1), adenylate kinase 1, and ADP-ribosylhydrolase like 1, associated with energy metabolism, also changed in this study. NAD-dependent GPD1 catalyzes the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate, while adenylate kinase 1 catalyzes the reaction from ATP and AMP to two ADP. The abundance of adenylate kinase1 gradually increased after 3 h postmortem, and the same enzyme also increased in intensity up to 72 h postmortem in a study of porcine muscle.2-4 Moreover adenylate kinase 1 was previously reported to be negatively correlated with drip loss.17 ADPribosylhydrolase like 1 is also a NAD-dependent enzyme and decreased in intensity postmortem. It has been reported that ADP-ribose can be removed from mono-ADP-ribosylated actin, and mono-ADP-ribosylation may control actin polymerization and depolymerization in animals.18 Cellular Defense and Stress. Several proteins that have protective functions changed significantly in abundance during the early postmortem storage period. Multiple spots representing the cytoplasmic protein DJ-1 all increased in abundance during postmortem storage. These are probably modifications of this enzyme. The intensities of all DJ-1 proteins increased

research articles to 10 h postmortem. The DJ-1 protein acts as a chaperone to protect neurons against oxidative stress and cell death.19 Antioxidant protein 2, SP-22 protein, and peroxiredoxin 2 belong to the peroxiredoxin family and have a cellular protective antioxidant activity by detoxifying peroxides.20 SP-22 protein was increased during the first hour after slaughter and then decreased in intensity. SP-22 was also reported to be decreased after 24 h postmortem storage in previous studies.5,6 It is a small (22 kDa) mitochondrial protein and has the ability to protect several free radical sensitive enzymes from inactivation by a metal-catalyzed, free-radical-generating system in addition to the antioxidant properties.21 Peroxiredoxin 2 is a cytosolic protein that is suggested to play a role in the signaling cascades of growth factors and switches from a peroxidase to a molecular chaperone at high H2O2 levels to inhibit apoptosis.22 Both peroxiredoxin 2 and antioxidant protein 2 increased in abundance up to 10 h and then slightly increased further until 24 h. Antioxidant protein 2 is the only 1-Cys member of the peroxiredoxin family, and this protein can also function as an antioxidant enzyme and therefore may play a role in the regulation of phospholipid turnover.23 Moreover, the other group of multiple spots was identified as HSPB1 protein (HSP27). The intensities are decreased 10 h after slaughter. The tendency of the expression profile of HSPB1 showed that the highest level of the protein was at 1 h after slaughter, and it then decreased. HSPB1 protein has several isoforms which may be caused by alternative splicing.24 HSPB1 protein has been suggested to protect actin filaments from damage and to prevent unfolded proteins from aggregation.25 However, the exact biochemical mechanism of HSPB1 is still unknown. Previous proteome studies in cattle5,6 and pork muscle2,4 also demonstrate that HSPB1 protein increases in intensity shortly after slaughter and decreases during storage. Ubiquitin-conjugating enzyme E2N increased in abundance up to 10 h and then decreased. Several reports show that increased proteolysis in skeletal muscle during the damage of cells was associated with an increase in the gene expression of this enzyme,26 suggesting that it might contribute to the survival of cells after damage. Biliverdin reductase B [flavin reductase (NADPH)] was clearly increasing in abundance postmortem. This enzyme can reduce biliverdin to bilirubin, which is the final product of heme catabolism and was more recently regarded as a potential physiological antioxidant in vivo.27 Thus, bilverdin reductase B is suggested to protect cells from oxidative damage and cell death.27 Postmortem metmyoglobin reduction is important for meat color, and this process needs NADH.28 The increasing level of biliverdin reductase B may lead to an increased amount of NADH and thereby contribute to beef color stability. The relationship of defense and stress proteins with meat quality traits is scarcely reported. It has been reported that postmortem changes of DJ-1 protein, HSPB1, substrate protein of mitochondrial ATP-dependent proteinase SP-22, and peroxiredoxin 2 are correlated to WB-shear force,17 drip loss,17 and meat color.15 Cell Structure. Tubulin, actin, and cofilin 2 all decreased in abundance postmortem. These proteins were also reported to change in a previous proteome study in cattle muscles.5 Tubulin is the major constituent of microtubules and is reported to have several types of post-translational modification, such as detyrosination, acetylation, phosphorylation, and polyglycylation, and these modifications play important roles in vivo.29 This may explain the occurrence of several spots Journal of Proteome Research • Vol. 6, No. 7, 2007 2729

research articles harboring tubulin on the 2-DE gels. Cofilin is a ubiquitous protein that binds to both G- and F-actin, and it has roles in stabilizing actin filaments and depolymerizing old actin filaments.30,31 Cofilin is previously shown to decrease in abundance 24 h postmortem in beef5 and pork.2,3 Proteasome. The proteasome 26S is one of the three most important proteolytic systems in skeletal muscle postmortem. It has been demonstrated that ATP is essential for keeping the 26S complex together, and the depletion of ATP will cause dissociation of the 26S complex into 20S complex and 19S complex.32 20S complex consists of multi-subunits with several functions, such as peptidylglutamyl peptide hydrolase, trypsinlike, and chymotrypsin-like activities.33 The 20S proteasome subunit was decreasing in this study, which confirms a previous postmortem proteome study in cattle muscles,5 suggesting rapid degradation of this enzyme postmortem. Earlier it has been reported that proteasomes are not involved in proteolysis of myofibrils.34,35 A recent study on bovine muscle reported that the 20S proteasome contributed to the degradation of the Z-line.36,37 Miscellaneous Proteins. The cytochrome bc1 complex, eukaryotic translation initiation factor 4AII, and growth factor receptor-bound protein 2 all decreased postmortem. Tumor protein (translationally controlled 1) increased in abundance up to 10 h and then decreased postmortem. The translationally controlled tumor protein38 is a calcium-binding protein that increased in abundance early postmortem, suggesting that this protein might be involved in the development of rigor mortis. Growth factor receptor-bound protein is involved in signaling pathways important for development of human breast cancer,39 while the cytochrome bc1 complex is a component in the mitochondrial respiratory chain, involved in the electrontransport process and ATP synthesis.40

Conclusions Proteomics is a powerful technology to study global changes of proteins occurring during postmortem storage. The proteins identified in this study are part of the biochemical network cooperating to prevent muscle cells from reducing the ATP level, stress, and cell death postmortem. Postmortem metabolism in muscle is not well understood. In this study, we investigated proteome changes in bovine longissimus thoracis muscle early postmortem. The results show that all identified metabolic enzymes are either involved in enzymatic reactions of the glycolysis and TCA pathway or associated with energy production. Several identified enzymes and proteins, namely, glycerol-3-phosphate dehydrogenase 1 (GPD1), ADP-ribosylhydrolase like 1, biliverdin reductase B, and cytochrome bc1 complex, are not directly involved in the glycolytic pathway but take part into the production of NAD+, which may further enter the glycolytic and TCA pathway to drive the synthesis of ATP. This indicates that the level of ATP is maintained for several hours after slaughter, and the energy production is still operative under the conversion of aerobic metabolism to anaerobic metabolism in muscle. Muscle cells are under stressful conditions after slaughter caused by nutrient and oxygen depletion. This is supported by the finding of stress and defense proteins, which were changed in abundance early postmortem. The protective functions of these proteins are probably to delay cell death, thus diminishing the impairment of stress. These changes could reflect important mechanisms related to development of a satisfactory meat quality. Additional 2730

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studies including more animals are required to reveal how these changes are related to meat quality traits in cattle.

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