Changes in Enzymes Associated with Energy Metabolism during the Early Post Mortem Period in Longissimus Thoracis Bovine Muscle Analyzed by Proteomics Xiaohong Jia,†,‡ Kjell I. Hildrum,† Frank Westad,† Eiliv Kummen,§ Laila Aass,# and Kristin Hollung*,† Matforsk AS, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, GENO Øyer, 2636 Øyer and Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O.Box 5003, N-1432 Ås, Norway Received March 27, 2006
Changes in metabolic protein levels in biopsies during the early post mortem period in the bovine longissimus thoracis muscle were investigated by 2-DE based proteome analyses. Nine NRF (Norwegian Red) dual purpose bulls were included in the study. Twenty-four proteins underwent changes between the two sampling times and were classified into two major groups: metabolic proteins and heat shock proteins. Of the metabolic proteins, 5 enzymes involved in the glycolytic pathway and the tricarboxylic acid (TCA) cycle, increased in intensities during the post mortem period. In addition, the NADPdependent enzyme 3-hydroxyisobutyrate dehydrogenase, associated with the TCA cycle in muscle, was increased. This documents that an increased aerobic energy metabolism occurs immediately after slaughter, with the aim to replenish the ATP levels in the muscle. Keywords: proteome analysis • bovine muscle • metabolic proteins • energy metabolism • 2-dimensional gel electrophoresis
Introduction Variability in meat quality traits is a major concern for both the meat industry and the consumer.1 Many factors can affect the final meat quality,2 of which the rate of early post mortem glycolysis seems to be an important determinant of eating quality.3,4 The rate and extent of the conversion of glucose to lactate is largely responsible for the pH decline post mortem and thus for the final meat quality.5 However, the biochemical mechanism behind the post mortem glycolysis in muscles, as well as the relationship between the rate of glycolysis and meat eating quality, are poorly understood. Soluble muscle proteins include many sarcoplasmic and mitochondrial enzymes, which are involved in the biochemical metabolic processes in the living animals. Although the in vivo roles of these metabolic proteins are well-known, their involvement in the change from aerobic metabolism in the living animal pre-slaughter to anaerobic metabolism post slaughter is not much investigated. The rate and extent of post mortem glycolysis is influenced by ATP turnover,5 which can be increased by i.e., electrical stimulation of the carcass. Hence, to turn the muscles into high quality meat, an improved * To whom correspondence should be addressed. Tel: +47 64970142. Fax: +47 64970333. E-mail:
[email protected]. † Matforsk AS. ‡ Department of Chemistry, Biotechnology and Food Science. § GENO Øyer. # Department of Animal and Aquacultural Sciences. 10.1021/pr060119s CCC: $33.50
2006 American Chemical Society
understanding of which metabolic proteins are involved in and how the biochemical and energy metabolism is regulated during the early post mortem period, is a prerequisite. Proteomics is an important cornerstone in post-genome sciences and has also been applied in meat science in recent years. For a general review, see Bendixen6 or more recent reports on proteomics in bovine muscles.6-9 2-DE based proteome analysis of porcine10,11 and cattle9 muscle showed that a wide range of metabolic proteins were changed during post mortem storage of meat. Most studies have so far been done on in vitro samples, partly because of the inherent problems in obtaining good in vivo biopsies. It is not unlikely that the in vivo analysis will, in general, yield the more relevant information, both with regard to interpretations of the genetic expression in the muscle and to post mortem events and meat quality parameters. The objective of this study was to investigate the changes in metabolic proteins in bovine muscles between pre and post mortem muscles.
Material and Methods Animals and Tissue Samples. The experiment included 9 NRF (Norwegian Red) dual-purpose young bulls (approximately 13 months of age/450 kg live weight) slaughtered from a performance test station (GENO-Breeding and AI Association) in 2004. During the test period (150-330 days of age), the bulls were kept in pens with 15-23 bulls/pen. The procedure for excision of biopsies had been approved by the Norwegian Journal of Proteome Research 2006, 5, 1763-1769
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research articles Animal Research Authority. Biopsies were obtained after sedation (xylasin 50-70 mg intra muscular) while the animals were in the feed rack 4 days before slaughter, basically as described.12 First, the local area on the hide was shaved and disinfected. A scalpel blade was used to cut an opening in the skin before a Bergstro¨m needle with inner diameter of 5 mm was inserted to collect the biopsy. Samples were collected from Longissimus thoracis at 6 cm depth cranial to the 12th rib. The biopsies were immediately frozen in liquid nitrogen and stored at -80 °C for later analysis. The bulls were transported (1 h) and slaughtered at a commercial slaughterhouse (Gilde HedOpp, Norway) immediately after arrival. Five of the nine carcasses were electrically stimulated (90 V) approximately 30 min post mortem. The samples from longissimus thoracis were removed, snap frozen in liquid nitrogen and stored at -80 °C approximately 45-60 min post mortem. Extraction of Proteins. Each biopsy was homogenized in 500 µL of TES buffer (10 mM Tris (pH 7.6), 1 mM EDTA and 0.25 M sucrose), and pieces of 200 mg frozen post mortem muscle tissue were homogenized in 1 mL of TES buffer by using ultraturrax (Polytron PT, Kinematica AG, Luzern Switzerland) at 12000 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 750 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.13 Homemade immobilized pH gradient (IPG) strips spanning the pH region 4-7 were used in the first dimension. For analytical 2-DE, 70 µg proteins were loaded on each IPG strip by in-gel rehydration, while 500 µg proteins were 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 and reaching a total of 70.000 Vh. Proteins were separated in 12% SDS-PAGE in the second dimension using the Ettan Dalt twelve Large Format Vertical System (Amersham Biosciences). A master gel for each muscle type was made from a mixture of all samples in the study. The analytical gels were stained by silver staining as described by Blum,14 while the preparative gels were stained according to Shevchenko.15 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 (Espon Expression 1680 Pro, Epson) with 8-bit color depth and a resolution of 240-dpi and analyzed using the ImageMaster 2D Platinum software Version 5.0 (Amersham Biosciences). The volume of the protein spot was calculated as the volume above the spot border, situated at 75% of the spot height (measured from the peak of the spot). The relative volume (%Vol) for each spot was calculated by dividing each spot volume by the total volume of all the spots in the 2-D gel. The spots were automatically matched with the spots of a master gel used as the reference gel. Artifacts and mismatch spots were removed manually. The protein spots were matched across all 36 gels (2 time points × 9 animals × 2 technical replicates) and the spot report was imported into Unscrambler version 9.2 (CAMO A/S, Norway) and Matlab version 7.0.4. Matched spots and sampling time (pre- and post-slaughter) were defined as x-variables and y-variables, respectively, in the 1764
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principle component analysis (PCA). PCA is a method to visualize the main variations and to detect clusters in a data population.16 Significant spots were identified by the cross-model validation (CMV, a two-layer cross-validation) variable selection method, which is based on partial least squares (PLS) regression with jack-knife estimates and t-tests.17 Spots with less than five matched proteins in the data table were excluded from the analysis. This resulted in a data set consisting of 690 protein spots and 670 protein spots for technical replicates 1 and 2, respectively. Matched spots (%Vol) and sampling time (pre and post slaughter) were defined as x-variables and y-variables, respectively, in the PLS analysis. All matched spots were analyzed with CMV18, where objects were left out in pairs (preand post-slaughter for each animal) while the rest of the objects were subject to cross-validation. The criterion for selecting variables is to select only the variables that are found significant in all individual cross-validations inside the CMV. Each technical replicate data set were analyzed individually and the reported significant spots (p < 0.05) are the protein spots found significant for both data sets. Protein Identification. The protein spots of interest were punched out of the preparative gels using pipet tips and extracted from gels according to the method of Jensen.19 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 speed-vac 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 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. A 30-µL portion trypsin digestion buffer (5 ng/µL) was added to the dried gel pieces, 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. 20 µL of the tryptic protein digests were loaded onto the column, and washed with 20 µL of 0.1% TFA. The peptides were eluted with 0.8 µL 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. 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 savitzky golay with 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 minimum three precursor ions recognized in the PMF search were performed to confirm the PMF-based protein identification.
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Figure 1. Silver-stained 2-DE master gel of bovine longissimus thoracis. A 50-µg portion of protein 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 in the post mortem samples are numbered.
The number of peptide matches, sequence coverage, molecular weight and pI were used to evaluate the database search results.
Results Changes in metabolic protein levels in biopsies from live animals to post mortem samples in the cattle longissimus thoracis muscle were investigated by 2-DE. Nine NRF (Norwegian Red) dual purpose bulls were included in the study and all samples were analyzed by technical replicates. Figure 1 shows a representative 2-DE pattern of the proteins extracted from muscle samples. The proteins in the molecular mass region of 10 kDa to 75 kDa, and the pH range between 4 and 7, were included in comparative analyses. Most of the soluble proteins were located within the range pH 5 to 7. Image analysis allowed matching of 833 spots across all 36 gels. Using PCA, principal components (PCs) were calculated and used to construct a coordinate system, in such a way that PC1 is the one that explain the most variation in the data and PC2 shows the second largest variation and so on.16 The resulting score plot allows easy interpretation of the main variation in the data set, and clusters of the samples are often visible using the first few PCs. The PCA score plot of the complete data set of spot intensities from comparative image analyses reveals that two clusters related to pre and post slaughter samples are formed by the first two components (Figure 2). In the score plot, preslaughter samples have negative scores and are located at the left side of the horizontal axis while post slaughter samples have positive scores and are at the right side of the horizontal axis.
Figure 2. Principal component analysis (PCA) score plot of 833 matching spots from bovine longissimus thoracis muscle at two sampling times. A1-A9: biopsies taken from nine animals. P1-P9: Samples taken after slaughter from the same nine animals.
PC1 and PC2 explain 13% and 11% of the total variance in the x-matrix, respectively. This moderate degree of explained variance of PC1 and PC2 is what usually can be expected for Journal of Proteome Research • Vol. 5, No. 7, 2006 1765
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Table 1. Differentially Expressed Proteins in Bovine Longissimus Thoracis Muscles Identified by MALDI-TOF-MS/MSa
spots
306 509 442 482 489 425 517 611 105 601 602 799 754
199
identified proteins
aldehyde dehydrogense family seven-member A1 (Antiquitin 1) phosphoglycerate kinase 1 enolase 3, beta muscle enolase 3, beta muscle enolase 3, beta muscle ATP-specific succinyl-CoA synthetase beta subunit isocitrate dehydrogenase 3 (NAD+) alpha 3-hydroxyisobutyrate dehydrogenase similar to hyaluronidase, partial CGI-105 protein CGI-105 protein similar to ubiquitin-conjugating enzyme E2N, partial substrate protein of mitochondrial ATP-dependent proteinase SP-22
686 698 699 706 763
similar to 60kDa heat shock protein, mitochondrial precursor heat shock 27kDa protein 1 heat shock 27kDa protein 1 heat shock 27kDa protein 1 heat shock 27kDa protein 1 crystallin, Alpha polypeptide 2
501 508 628
tropomyosin 1 tropomyosin 1 proteasome activator 28 alpha subunit
matched peptides/ sequence coverage
NCBI or SWISS-PROT accession no. (source)
theoretical pI/Mr
fold changeb
Metabolic Proteins 5.70/56000 13/28%
gi|25108887 (Human)
6.24/55845
+1.34
6.20/39000 6.10/47000 6.25/40000 6.60/40000 5.85/47000
11/43% 16/41% 16/39% 17/44% 10/26%
gi|56757507 (Pig) gi|6679651 (Mouse) gi|6679651 (Mouse) gi|6679651 (Mouse) gi|3766197 (Human)
8.02/44957 6.73/47337 6.73/47337 6.73/47337 5.84/46732
+1.67 +2.43 c -3.98 +1.68
6.45/38000
15/49%
gi|27807161 (Bovine)
6.76/40098
+1.66
5.60/32000
9/31%
Gi|76615500 (Bovine)
5.54/32059
+2.15
6.20/75000 5.90/34000 6.20/34000 5.50/16500
11/16% 9/36% 14/47% 10/63%
gi|61884971 (Bovine) gi|37780029 (Bovine) gi|37780029 (Bovine) gi|61852792 (Bovine)
7.89/73370 7.00/34861 7.00/34861 5.19/16048
-1.80 +1.97 +1.58 +1.51
6.20/22000
8/38%
gi|627764 (Bovine)
5.73/21709
+1.74
gi|61815075 (Bovine)
5.93/63786
+1.32
gi|61553385 (Bovine) gi|61553385 (Bovine) gi|61553385 (Bovine) gi|61553385 (Bovine) gi|27805849 (Bovine)
6.49/17602 6.49/17602 6.49/17602 6.49/17602 6.76/20024
+3.38 +5.76 +5.05 +4.58 +7.22
gi|57281697 (Bovine) gi|57281697 (Bovine) gi|66792738 (Bovine)
4.69/32732 4.69/32732 5.78/28816
-6.70 -6.47 +1.50
experimental pI/Mr
Stress Proteins 5.25/65000 20/40% 5.60/25000 12/72% 5.45/25000 10/60% 5.50/25000 8/58% 5.95/25000 7/50% 6.90/20000 12/58% Unclassified Proteins 4.60/37000 17/47% 4.60/37000 17/47% 5.80/29000 15/52%
a Of the 24 spots changed in the pre and post slaughter samples with 5% significance level, we identified 22 spots. 2 spots could not be identified. b Fold change: the expression ratios between the means of spot volumes (% Vol) at two sampling times resulting from digital image analysis. + Indicates increased protein intensity and - indicates decreased protein intensity after slaughter. c The spots were detected only in the biopsies.
2-DE data due to the high noise-level in the gel images. In this study, we used CMV for selection of protein spots changing after slaughter. In the first set of technical replicates 49 protein spots were significantly changed, and in the second technical replicate 56 proteins were significantly changed. The 24 protein spots numbered in Figure 1 were significantly changed in both data sets and were selected for protein identification. Among the selected 24 protein spots, 18 spots increased and 6 decreased in intensities in the post mortem samples, respectively. A total of 22 spots were successfully identified by MALDITOF-MS/MS (Table 1). Two protein spots were not appearing in the preparative gels and were therefore not identified. In this study, 16 spots were identified by matching peptide data to bovine protein sequences in the database, while the other 6 spots were identified due to interspecies homology to human and mouse protein sequences. For some protein spots a shift in molecular weight (spots 482, 489, 509, 706, 698, 686, and 699) and pI value (spots 105, 509, 601, 602, 482, 489, 442, 706, 698, 686, and 699) compared to the theoretical pI and MW were observed. Multiple protein spots representing the same protein were observed for spots 699, 706, 698, and 686, which are all identified as HSP27. These have the same MW but different pI. The expression ratios (fold change) from the image analysis indicated that HSP27KDa protein (spots 698 and 699) and Crystallin, alpha polypeptide 2 (spot 763) were markedly increased after slaughter. Furthermore, protein spots 601 and 1766
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602 were identified as CGI-105 protein with the same MW but different pI. Protein spots 482, 489, and 442 all harbor enolase 3 and they differ in both MW and pI. The identified proteins can be divided into three groups, metabolic proteins, stress proteins and unclassified proteins. Of the metabolic proteins 6 enzymes increased in intensities post mortem. These were enolase 3 (spot 442), aldehyde dehydrogenase family seven-member A1 (spot 306), phosphoglycerate kinase 1 (spot 509), ATP-specific succinyl-CoA synthetase beta subunit (spot 425) and isocitrate dehydrogenase 3 (NAD+) alpha (spot 517). All these enzymes are involved in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle. Moreover, a NADP-dependent -metabolic protein, 3-hydroxyisobutyrate dehydrogenase, increased in intensity.
Discussion By using 2-DE based proteomics, we were able to study shifts in energy metabolism at the protein level in bovine muscle from the in vivo state to shortly after slaughter of the animals. In this study, several glycolytic enzymes involved in energy metabolism, in addition to 3-hydroxyisobutyrate dehydrogenase and two enzymes in the TCA cycle, increased in intensity post mortem (Figure 3). Phosphoglycerate kinase which catalyses the formation of 3-phosphoglycerate,20 increased in intensity. The other glycolytic enzyme, enolase 3, was identified in three spots, where one (spot 442) increased and two (spots 482 and 489) decreased in intensities. However, the enolase 3
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Figure 3. Biochemical pathways of the energy metabolism that are influenced in bovine longissimus thoracis post mortem. The identified metabolic enzymes by MALDI-TOF-MS/MS are shown in red. The arrows in blue show increased enzyme abundance.
proteins have different pI, probably due to posttranslational modification or proteolysis. The finding that several glycolytic enzymes increased in intensities after slaughter may suggest increased rate of glycolysis, to support and maintain the ATP production. In muscles, energy is stored mainly as creatine phosphate and glycogen. As some of the carcasses were electrically stimulated prior to the collection of the post mortem samples, the ATP reserves in the muscles were most likely depleted by the energy demand of the provoked muscle contraction during electrical stimulation. However, degradation of the glycogen stores via the glycolytic pathway is a major source of ATP replenishment. This was supported by the finding that three of the enzymes involved in glycolysis increased in abundance in the samples taken post slaughter, compared to samples from the live animals. As long as oxygen is available, the resulting pyruvate from the glycolysis will be used as substrate for the TCA cycle. In this study, two enzymes in the TCA cycle, namely, ATP-specific succinyl-CoA synthetase beta subunit (spot 425) and isocitrate dehydrogenase 3 (NAD+) alpha (spot 517) increased in abundance. In addition, a NADP-dependent 3-hydroxyisobutyrate
dehydrogenase, which is involved in the degradation of a glucogenic amino acid (Valine) to produce the metabolic intermediate (succinyl-CoA) that can be further oxidized by the TCA cycle,21 increased in abundance. This suggests that the oxidative metabolism was still operative and even maybe at a higher rate compared to the situation in the live animals. After bleeding of the animals, the circulatory oxygen supply ceases. At the time of post mortem sampling, only limited amounts of blood will be present in the muscle, and oxygen supply from blood is probably negligible. However, the muscles contain oxygen bound to myoglobin, which will be released upon oxygen depletion to support oxidative metabolism.5 As long as the oxygen supply is above a threshold level, the pyruvate is converted to energy via the TCA cycle. However, when the oxygen supply ceases, pyruvate is converted to lactate, and pH in the muscle drops. The increased expression of these metabolic enzymes may have effects on the rate of glycolysis and pH decline, which in turn could influence the final eating quality. Previous studies on bovine muscles showed that the rate of pH decline influences the tenderness of beef after aging.3,4 Work is in progress to elucidate if the level of these enzymes will have an effect on tenderization of beef and also Journal of Proteome Research • Vol. 5, No. 7, 2006 1767
research articles on other meat quality parameters. It is possible that the extent of the shift in energy metabolism could influence the rate of pH decline post mortem, and thereby explain some of the variation in final tenderness of beef. In a corresponding proteome study of porcine muscles during early post mortem storage of meat, enolase 3 and phosphoglycerate kinase were found to increase up to 72 h post mortem.10,11 Moreover, a recent study on porcine muscle reported that the increasing juiciness and paleness of pork is highly related to denaturation of several sarcoplasmic proteins in the muscle cell.22 Proteins with other metabolic functions were also changed post mortem in this study. Hyaluronidase is responsible for degrading hyaluronic acid in the extracellular matrix, which is known to interact with the large proteoglycans that forms the structural network of the extracellular matrix in the connective tissues in muscles.23 A breakdown of this extracellular matrix may activate the proteolytic system and thereby promote enzymatic degradation.24 The observed decrease in intensity of hyaluronidase may indicate that it is rapidly proteolyzed caused by depletion of ATP. The role of CGI-105 protein in muscle metabolism is unknown. In the present study, the stress proteins Crystallin (Rpolypeptide 2), HSP27 and HSP60 were increased shortly after slaughter. Crystallin (R-polypeptide 2) belongs to the small heat shock protein (HSP20) family,25 as well as HSP27 and HSP60, which prevent degradation and structure damage of proteins from apoptotic processes in muscle cells.26 The observed multiple spots of HSP27 were most probably due to modifications or different isoforms of the protein. Phosphorylations of the N terminus of HSP27 is coupled to chaperone activity in cells.27 In a previous study of post mortem storage of bovine muscle we observed a decrease of HSP27.9 HSP27 and Crystallin (R-polypeptide) are believed to be involved in stabilization and regulation of the myofibrillar proteins and to protect actin filaments and other cytoskeletal proteins from fragmentation caused by stress conditions.28,29 Hence, these stress proteins might be the potential markers of the change of myofibril structure in muscle post mortem. In addition, ubiquitinconjugating enzyme E2N (spot 799) and SP-22 (spot 754), which also have defense functions in the muscle cell, were increased in this study. Ubiquitin-conjugating enzyme E2N is known to play a role in the cell cycle control and differentiation.30 The increased level of this enzyme may suggest that it contributes to the survival of cells after slaughter. SP-22 protect enzymes from oxidative damage in the mitochondria,31 and it was also changed in porcine and bovine muscles post mortem.9,32 Hence, the increased expression level of these proteins might act as defense systems in the muscle cells immediately after slaughter. One proteasome subunit increased in intensity immediately after slaughter. It is well-known that the proteasome complex is strictly ATP dependent, and the depletion of ATP after slaughter may cause rapid dissociation of the proteasome complex into multiple subunits.33 Thus, it was not surprising that the proteasome subunits increased in intensity immediately after slaughter. This was also observed in the previous study of bovine muscle.9 Proteasomes are also reported to play a role in synergy with other proteolytic enzymes during aging of bovine muscle,34 however, it is unclear whether the proteasome complex is of importance with regard to meat quality traits. The goal of this study was to investigate the changes in metabolic proteins from live animals to samples immediately 1768
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after slaughter. Several processes such as transportation, lairage, stunning, exsanguination and dehiding could contribute to the changes in the protein profile. Thus, the observed changes in the present study was the consequence of the total of all these processes. Previously, it has been demonstrated that electrical stimulation increases the rate of post mortem glycolysis,35 and it is therefore widely used in technological processing of meat to improve tenderness. However, we believe that the identified changes are not the result of electrical stimulation itself as both stimulated and unstimulated samples were included in this study. In this study, we did not address these questions, but rather investigated the consequence of all these processing parameters. Further studies are needed to separate the individual effects of the processing steps on the protein profile. We are currently in progress to elucidate the effect of electrical stimulation on the proteome of bovine muscles.
Conclusion We observed a clear shift in energy metabolism in the muscle post mortem with an increase in enzymes involved in both the glycolytic pathway as well as in the TCA cycle. These findings suggest that an increased aerobic energy metabolism occurs the first hour after slaughter. The increased aerobic energy metabolism will probably affect the rate of glycolysis in muscles after slaughter and eventually lead to variation of meat quality. However, additional studies including more animals are required to reveal how changes in protein expression in pre and post mortem muscles are related to meat quality traits in cattle.
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Journal of Proteome Research • Vol. 5, No. 7, 2006 1769
