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Journal of Animal Science - Symposia

2013 EARLY CAREER ACHIEVEMENT AWARD— Proteomics of muscle- and species-specificity in meat color stability12


This article in JAS

  1. Vol. 92 No. 3, p. 875-882
    Received: Oct 18, 2013
    Accepted: Dec 31, 2013
    Published: November 24, 2014

    3 Corresponding author(s):

  1. S. P. Suman 3,
  2. G. Rentfrow*,
  3. M. N. Nair* and
  4. P. Joseph
  1. Department of Animal and Food Sciences, University of Kentucky, Lexington 40546
    Kalsec, Inc., Kalamazoo, MI 49005


Meat color is the most important quality trait influencing consumer purchase decisions. The interinfluential interactions between myoglobin and biomolecules govern color stability in meat. The advances in proteomics, such as high throughput analytical tools in mass spectrometry, 2-dimensional electrophoresis, and bioinformatics, offer themselves as robust techniques to characterize the proteome basis of muscle- and species-specific meat color phenomena. Differential abundance of chaperones and antioxidant proteins contributes to muscle-specific color stability in beef; the greater abundance of chaperones and antioxidant proteins in color-stable Longissimus lumborum than in color-labile Psoas major protects myoglobin and contributes to superior color stability of beef Longissimus steaks. Lipid oxidation-induced myoglobin oxidation is more critical to beef color than pork color due to the inherent differences in myoglobin chemistry; the number of nucleophilic histidine residues adducted by reactive aldehydes is greater in beef myoglobin than in pork myoglobin. Preferential adduction of secondary products of lipid oxidation to beef myoglobin accelerates metmyoglobin formation at a greater degree than in its pork counterpart. Mass spectrometric investigations revealed that although cherry-red carboxymyoglobin is more stable than oxymyoglobin, both redox forms undergo lipid oxidation-induced oxidation in model systems. The accuracy of mass spectrometry to detect the molecular mass of proteins has been applied to differentiate myoglobins from closely related meat animals, such as goats and sheep or emu and ostrich. In addition, this approach indicated that turkey myoglobin is 350 Da greater in molecular mass than beef myoglobin, and the unique biochemistry of turkey myoglobin could be responsible for its greater thermostability in model systems as well as the pink color defect observed in fully cooked uncured turkey products.


At the point-of-sale, color of fresh red meats is an important quality trait governing purchase decisions (Mancini and Hunt, 2005). Consumer-desirable cherry-red color is critical to meat retailing, and any deviation from this color is considered by buyers as an indication of spoilage and unwholesomeness, leading to product rejection. Discolored fresh meat cuts are often discarded, sold at discounted price, or ground to low-value products before their microbial quality is compromised; these retail practices not only incur revenue loss but also can impact sustainability of animal agriculture (Troy and Kerry, 2010). It has been estimated that discoloration-induced quality deterioration results in more than $1 billion annual revenue loss to the U.S. meat industry (Smith et al., 2000). Chemistry of myoglobin and its interactions with other biomolecules (i.e., ligands, aldehydes, and proteins) influence color stability in postmortem skeletal muscles (Suman and Joseph, 2013). Previous research documented the role of several intrinsic (i.e., sarcoplasmic proteins, lipid oxidation, amino acid sequence) and extrinsic (i.e., ligands, antioxidants) factors on myoglobin redox chemistry, and elucidating the molecular basis of these interactions is critical to developing strategies to improve meat color stability (Faustman et al., 2010).


The proteome is the protein counterpart of the genome, and it is defined as the set of all proteins expressed in a cell, tissue, organ, or organism at a specific point of time (Liebler, 2002). Whereas the genome is static, the proteome is dynamic and changes over time (Peng and Gygi, 2001). For instance, the skeletal muscle proteome of an animal undergoes constant changes during its life (Ohlendieck, 2011), and the muscle proteome of a newborn animal is significantly different from a full-grown adult animal ready to be harvested. Furthermore, the proteome continues to change in postmortem skeletal muscles during storage and aging (Hollung et al., 2007).

Proteomics is the systematic evaluation of the proteome for identification, quantification, and functional characterization of proteins (Liebler, 2002). Successful analysis of the proteome is, in general, based on the following four major scientific platforms: 1) simple purification of proteins from complex biological systems, 2) accurate methods to harvest data on proteins, 3) access to robust protein databases, and 4) utilization of algorithms to generate structural and functional information on the proteins (Gevaert and Vandekerckhove, 2000). In the modern-day life sciences, 2-dimensional gel electrophoresis has replaced 1-dimensional SDS PAGE as the choice of technique for protein separation. Two-dimensional gel electrophoresis allows for simultaneous separation of several thousands of proteins based on their charge as well as their mass. In the postgenomic era, mass spectrometry is an important tool to correlate proteins with their genes (Yates, 1998). Noticeably, the last few decades witnessed the emergence of mass spectrometry from the technique for primarily analyzing volatile compounds to a robust tool to evaluate macromolecules such as proteins and peptides. The seamless integration of HPLC and 2-dimensional gel electrophoresis with mass spectrometry and ever-expanding protein databases has enabled life scientists to rapidly separate, identify, and quantify several thousand proteins and their modifications in biological systems.


The applications of proteomic techniques have grown exponentially in the agricultural and life sciences, whereas their applications in meat science are in relatively early stages. Proteomic tools have been applied in pre-harvest as well as post-harvest aspects of meat production (Bendixen 2005; Mullen et al., 2006). Pre-harvest applications explain biochemistry of food animal growth (Doherty et al., 2004) and muscle biology (Okumura et al., 2005), whereas postharvest aspects focus on biochemical mechanisms governing the conversion of muscle to meat (Lametsch et al., 2002), tenderness (Laville et al., 2009), and color (Sayd et al., 2006). For studying the proteome basis of meat tenderness and muscle-to-meat conversion, the main strategy exploited by investigators is the evaluation of whole-muscle proteome or its fractions (Huff-Lonergan et al., 2011; Lametsch 2011). On the other hand, investigations on proteomics of meat color focused on the interactions between myoglobin and biomolecules.


This paper provides an overview of our research applying proteomics and mass spectrometry in attempt to explain how and why certain color phenomena happen in meats from livestock and poultry. The foci of these investigations were muscle-specific nature of beef color, species differentiation of myoglobins, species-specificity in lipid oxidation-induced discoloration, and pink color defect in cooked turkey.

Muscle-Specificity in Beef Color

Among the multitude of endogenous factors governing beef color stability, muscle-specificity received significant attention with the successful completion of muscle profiling (Von Seggern et al., 2005) and due to the increasing popularity of whole-muscle cuts in retail markets. Individual muscles in live animals have specific anatomical locations as well as physiological functions, resulting in differences in biochemistry, ultrastructure, and metabolism. As a result, each beef muscle demonstrates unique postmortem color stability and biochemistry (Hunt and Hedrick, 1977). Several researchers (McKenna et al., 2005; Seyfert et al., 2006) documented that myoglobin oxidation and surface discoloration in beef cuts are muscle-dependent. Furthermore, based on color stability attributes, muscles in a beef carcass are categorized as color-stable and color-labile. Muscles exhibiting low metmyoglobin reduction (Ledward, 1985) and high oxygen consumption (O’Keeffe and Hood, 1982) are defined as color-labile, whereas those with increased reducing activities are color-stable (Reddy and Carpenter, 1991).

In meat color research, two major beef muscles extensively utilized to investigate muscle-specificity are Psoas major and Longissimus lumborum. Psoas major, the muscle marketed as filet mignon or tenderloin, is color-labile, whereas Longissimus lumborum, retailed as New York Strip steak, is a color-stable muscle (O’Keeffe and Hood, 1982; McKenna et al., 2005; Seyfert et al., 2006). Earlier studies documented that these two beef muscles responded differentially to the common packaging systems used in the U.S. for retailing (Fig. 1; Mancini et al., 2009) and demonstrated differences in internal cooked color (Fig. 2; Suman et al., 2009b). Previous investigations to explain the fundamental basis of muscle-specificity of beef color focused on muscle biology and biochemical attributes. Psoas major demonstrated greater lipid oxidation, less metmyoglobin reducing activity, and less color stability than Longissimus lumborum (McKenna et al., 2005; Seyfert et al., 2006). Other studies attributed the muscle-specific nature of beef discoloration to various enzymes, such as cytochrome b5 (Arihara et al., 1995), metmyoglobin reductase (Hagler et al., 1979), and glutathione peroxidase (Daun et al., 2001). Noticeably, these investigations focused on few critical enzymes or general biochemical changes in the muscle food matrix.

Figure 1.
Figure 1.

Surface a* value (redness) of raw beef Longissimus lumborum (LL) and Psoas major (PM) steaks under different packaging systems after 9 d of refrigerated storage. Means with a different letter differ (P < 0.05). Reproduced from Mancini et al. (2009) with permission of Elsevier Limited, Oxford, United Kingdom.

Figure 2.
Figure 2.

Internal a* value (redness) of beef Longissimus lumborum (LL) and Psoas major (PM) steaks cooked after refrigerated storage. Means with a different letter differ (P < 0.05). Reproduced from Suman et al. (2009b) with permission of Elsevier Limited, Oxford, United Kingdom.


The aforementioned studies established a sound foundation, and we have utilized tools in proteomics and mass spectrometry to characterize the roles of sarcoplasmic proteome components in differential color stability of beef muscles. Sarcoplasmic proteome accounts for approximately one-third of the total proteins in skeletal muscles and comprises soluble proteins and enzymes interacting with myoglobin (Scopes, 1970). Using 2-dimensional electrophoresis and tandem mass spectrometry, we evaluated the sarcoplasmic proteome of Psoas major and Longissimus lumborum to characterize the differential abundance of proteome and its relationship with color attributes of beef retailed in aerobic packaging (Joseph et al., 2012b). Analyses of sarcoplasmic proteome identified 16 differentially abundant proteins in Psoas major and Longissimus lumborum, and the identified proteins included antioxidant proteins (i.e., thioredoxin, peroxiredoxin-2, dihydropteridine reductase), chaperones (i.e., heat shock protein-70 kDa, heat shock protein-27 kDa, and stress-induced phosphoprotein 1), and enzymes involved in energy metabolism (i.e., β-enolase, triose phosphate isomerase, pyruvate dehydrogenase, creatine kinase). Statistical analyses revealed that Longissimus lumborum exhibited an overabundance of proteins positively correlated to surface redness (i.e., aldose reductase, creatine kinase, and β-enolase) and color stability (i.e., peroxiredoxin-2, peptide methionine sulfoxide reductase, and heat shock protein-27 kDa), whereas Psoas major demonstrated overabundance of mitochondrial aconitase that was negatively correlated with redness (Table 1). Antioxidant proteins (i.e., superoxide dismutase, catalase, and glutathione peroxidase) can minimize meat discoloration by preventing metmyoglobin formation and lipid oxidation (Decker et al., 2000); lipid oxidation accelerates myoglobin oxidation and discoloration in fresh red meats (Faustman et al., 2010). Chaperones (heat shock protein-27 kDa and αB-crystallin) prevent protein aggregation and denaturation, which could compromise myoglobin stability and meat color (Sayd et al., 2006). Our study concluded that the superior color stability of beef Longissimus lumborum could be attributed to the overabundance of antioxidant proteins and chaperones. Furthermore, these findings underscored the necessity to develop muscle-specific processing (e.g., packaging, enhancement, and antioxidant) strategies to improve color stability of retail fresh beef.

View Full Table | Close Full ViewTable 1.

Functional roles of differentially-abundant sarcoplasmic proteins in beef Longissimus lumborum (LL) and Psoas major (PM) muscles and the correlation of differentially abundant proteins with color attributes1

Protein Functional category Overabundant in muscle Color trait Correlation coefficient
Aldose reductase Enzyme LL a* value2 + 0.64
Creatine kinase Enzyme LL a* value + 0.72
β-enolase Enzyme LL a* value + 0.64
Pyruvate dehydrogenase Enzyme LL a* value + 0.65
Pyruvate dehydrogenase Enzyme LL R630/5803 + 0.67
Peroxiredoxin-2 Antioxidant LL R630/580 + 0.92
Heat shock protein-27 kDa Chaperone LL R630/580 + 0.87
Peptide methionine sulfoxide reductase Enzyme LL R630/580 + 0.88
Stress-induced phosphoprotein 1 Chaperone LL R630/580 + 0.75
Peptide methionine sulfoxide reductase Enzyme LL MRA4 + 0.63
Mitochondrial aconitase 2 Enzyme PM a* value – 0.59
1Reproduced from Joseph et al. (2012b) with permission of American Chemical Society, Washington, DC.
2a* value = Redness.
3R630/580 = Ratio of reflectance at 630 nm and at 580 nm; greater value indicates lower metmyoglobin content and thus greater color stability.
4MRA = Metmyoglobin reducing activity.

Species-Specificity in Lipid Oxidation-Induced Meat Discoloration

The cellular mechanisms responsible for preventing oxidative damage of macromolecules cease to exist shortly after death of the animal, whereas the oxidative reactions in skeletal muscles continue postmortem (Lund et al., 2011; Zhang et al., 2013). In the complex muscle food matrix, oxidation of lipids and myoglobin are interinfluential (Baron and Andersen, 2002; Faustman et al., 2010); oxidation of one favors the other resulting in deterioration of flavor as well as color quality. The contribution of lipid oxidation products (such as aldehydes) to rancidity development in meats has been well known for several decades, whereas their role in off-color phenomenon is relatively new. The developments in mass spectrometry have enabled elucidating the mechanistic interactions between reactive products of lipid oxidation and myoglobin leading to meat discoloration. Research in last two decades provided critical evidence that the covalent modification of histidine residues (via Michael addition) in myoglobins of horse (Faustman et al., 1999), pork (Lee et al., 2003), and beef (Alderton et al., 2003) by reactive aldehydes is responsible for lipid oxidation-induced meat discoloration. The aforementioned studies reported that adduction of aldehydes at the distal (i.e., position 64) and proximal (i.e., position 93) histidines, which are critical to myoglobin functionality (Cornforth and Jayasingh, 2004), compromises the heme protein’s redox stability and accelerates formation of brown metmyoglobin.

Lipid oxidation and subsequent discoloration in meat can be prevented by the use of antioxidants in animal feeds, and vitamin E (α-tocopherol) is one among them. Lipid-soluble vitamin E is an effective polyphenolic free radical scavenger that inhibits peroxidation of polyunsaturated fatty acids in cell membranes (Buttriss and Diplock, 1988), and the increased tissue levels of vitamin E protect not only membrane lipids but also myoglobin from oxidation. Supranutritional supplementation of vitamin E in the finishing diet of cattle increased color and lipid stability of beef (Faustman et al., 1989; Lynch et al., 1999). However, in vitamin E–supplemented pork, although lipid oxidation was minimized significantly, a color-stabilizing effect was not readily observed (Lanari et al., 1995; Phillips et al., 2001). Pork muscles generally have a greater proportion of unsaturated fatty acids than beef. Logically, one would anticipate that pork lipids, being more unsaturated, would undergo lipid oxidation more readily and produce more secondary products than beef lipids. In turn, this would result in a reduction in color stability in pork, which could be prevented by vitamin E. However, findings that pork did not demonstrate improved color stability from vitamin E supplementation indicated that the susceptibility of pork myoglobin to lipid oxidation is different from that of beef myoglobin.

In concurrence with previous studies on lipid oxidation-induced myoglobin oxidation, we employed 4-hydroxynonenal (HNE) as a model aldehyde to explain species-specific nature of this phenomenon in beef and pork (Suman et al., 2006a, 2007). Our investigation (Suman et al., 2006a) examined the fundamental basis of previously reported differences in pork and beef color stability in the presence of lipid oxidation. This study observed that at meat conditions (i.e., pH 5.6 and 4°C), pork myoglobin is less susceptible to aldehyde adduction than its beef counterpart. Mass spectrometric analyses indicated that while pork myoglobin formed monoadducts with HNE after 3 d incubation at meat conditions, beef myoglobin formed both monoadducts and diadducts. Tandem mass spectrometry identified 2 histidine residues in pork myoglobin adducted by HNE, whereas 4 histidines were adducted in beef myoglobin. These results indicated that the amino acid sequence of beef myoglobin predisposes it to nucleophilic attack by reactive lipid oxidation products at a greater degree than in pork myoglobin.

Through a quantitative proteomics approach, further investigations in this area (Suman et al., 2007) characterized the kinetics of aldehyde adduction in beef and pork myoglobins. In general, mass spectrometry is used as a qualitative tool rather than a quantitative one in proteomic research because of the unpredictable ionization properties of peptides and proteins (Liebler, 2002). Mason and Liebler (2003) developed a technique using isotope-coded phenyl isocyanate for labelling peptides and subsequently quantifying the abundance of their daughter ions through tandem mass spectrometry. Employing this strategy, HNE-adducted myoglobin peptides were labelled and quantified (Suman et al., 2007); this approach determined that in pork myoglobin, histidine 36 was the most reactive to HNE, whereas in beef myoglobin, histidines 88 and 81 were the readily adducted ones. These findings indicated that preferential adduction of aldehydes to histidines 88 and 81 can change the 3-dimensional structure of beef myoglobin, thereby exposing the heme accessible to the prooxidants and thus leading to meat discoloration. Furthermore, the aforementioned investigations (Suman et al., 2006a, 2007) demonstrated that lipid oxidation is more critical to color stability in beef than in pork and explained why dietary vitamin E did not improve color stability in pork.

Emu and ostrich are sources of nutrient-rich exotic meats (Sales and Horbanczuk, 1998; Andrews et al., 2000). Fresh meat from these ratite birds is dark red in color, similar to red meats harvested from mammalian livestock (Morris et al., 1995; Sales, 2007). The abundance of polyunsaturated fatty acids makes ratite meats highly susceptible to lipid oxidation (Sales and Oliver-Lyons, 1996; Horbanczuk et al., 1998), which in turn can compromise color stability (Faustman et al., 2010). The molecular basis of lipid oxidation-induced oxidation in emu and ostrich myoglobins was characterized, in comparison with beef myoglobin, using mass spectrometry (Nair et al., 2014). At physiological condition (i.e., pH 7.4 and 37°C), HNE-induced metmyoglobin formation was greater in ostrich oxymyoglobin than in emu and beef oxymyoglobins. Monoadducts were detected in both emu and ostrich myoglobins after 6-h incubation with HNE. Tandem mass spectrometry revealed that histidine 36 was adducted by HNE in ostrich myoglobin, whereas histidines 34 and 36 were adducted by HNE in emu myoglobin. Nonetheless, simultaneous adduction at positions 34 and 36 was not observed in emu myoglobin, possibly due to steric hindrance. Although emu and ostrich myoglobins share 95% sequence similarity, they exhibited differences in molecular interactions with secondary products of lipid oxidation. These results indicated that variations in the primary structure in avian myoglobins can influence the heme protein’s susceptibility to lipid oxidation-induced oxidation.

The mass spectrometric studies on lipid oxidation-induced meat discoloration were accomplished using oxymyoglobin, which is the cherry-red redox form responsible for the color of conventionally bloomed fresh red meats and of those retailed in high-oxygen-modified atmosphere packaging. Carbon monoxide binds strongly to myoglobin to form carboxymyoglobin, which also has a bright cherry-red color and is very stable against oxidation (Sorheim et al., 1999). The U.S. Food and Drug Administration approved 0.4% carbon monoxide in modified atmosphere packaging for retailing red meats in 2004 (Eilert, 2005). Thus, carboxymyoglobin also became relevant to the U.S. meat industry (Cornforth and Hunt, 2008). Applied aspects of carbon monoxide–modified atmosphere packaging were extensively investigated, whereas fundamental concepts of carboxymyoglobin chemistry were not completely understood (Mancini and Hunt, 2005). Of specific interest is the redox stability of carboxymyoglobin in the presence of prooxidant products of lipid oxidation. Preliminary investigation (Suman et al., 2006b) indicated that carboxymyoglobin undergoes lipid oxidation-induced browning. In further studies, we have utilized mass spectrometry to characterize the molecular basis of lipid oxidation-induced browning in equine carboxymyoglobin (Joseph et al., 2009) and observed that HNE formed adducts with carboxymyoglobin. Furthermore, HNE adduction in carboxymyoglobin was pH- and temperature-dependent in nature. Mass spectra revealed that HNE formed monoadducts, diadducts, and triadducts with carboxymyoglobin (Fig. 3) at physiological conditions (pH 7.4 and 37°C), whereas monoadducts and diadducts were formed (Fig. 4) at meat storage conditions (pH 5.6 and 4°C). These results are similar to those previously reported in oxymyoglobin (Alderton et al., 2003; Suman et al., 2007). The protonation of the imidazole group at pH 5.6 decreases its nucleophilicity and, thus, render histidines unfavorable residues for HNE adduction. Our further studies exploited tandem mass spectrometry and documented that histidines at positions 24, 36, 48, 81, and 93 in equine carboxymyoglobin were adducted by HNE (Joseph et al., 2010c).

Figure 3.
Figure 3.

Mass spectrum of carboxymyoglobin (COMb) incubated with 4-hydroxynonenal (HNE) at pH 7.4 and 37°C for 6 h. Reproduced from Joseph et al. (2009) with permission of Elsevier Limited, Oxford, United Kingdom. See online version for figure in color.

Figure 4.
Figure 4.

Mass spectrum of carboxymyoglobin (COMb) incubated with 4-hydroxynonenal (HNE) at pH 5.6 and 4°C for 7 d. Reproduced from Joseph et al. (2009) with permission of Elsevier Limited, Oxford, United Kingdom. See online version for figure in color.


Myoglobin Species Differentiation

Color phenomena in conventional livestock and poultry have been extensively studied, whereas the biochemistry of color in exotic and emerging meat animals received far less attention. Investigations in our laboratory successfully employed mass spectrometry, coupled with Edman degradation, to characterize the chemistry of myoglobins from exotic as well as emerging meat animals. The amino acid sequences of goat (Suman et al., 2009a), emu (Suman et al., 2010), American bison (Joseph et al., 2010b), and white-tailed deer (Joseph et al., 2012a) myoglobins were determined. The findings indicated that goat myoglobin differs from its sheep counterpart at 2 amino acids (i.e., at positions 8 and 52; Suman et al., 2009a), whereas emu myoglobin demonstrated differences at 8 residues compared with ostrich myoglobin (Suman et al., 2010). On the other hand, myoglobins of several closely related ruminants were found to share 100% similarities in primary structure, for instance, bison and beef (Joseph et al., 2010b) and white-tailed deer and red deer (Joseph et al., 2012a).

Pink Color Defect in Turkey

Pink color defect is the pinkish appearance in fully cooked and uncured turkey. Consumers consider such products uncooked leading to rejection (Holownia et al., 2003). Although previous studies insinuated possible unique biochemistry of turkey myoglobin as the underlying reason for pink color defect (Cornish and Froning, 1974; Trout, 1989), efforts were not streamlined in a direction to elucidate the molecular mechanism. Research in our laboratory characterized turkey myoglobin and its physicochemical properties in comparison with beef myoglobin to explain this phenomenon (Joseph et al., 2010a, 2011). Turkey myoglobin demonstrated greater thermostability than beef myoglobin in model systems simulating typical meat cooking conditions (Joseph et al., 2010a), and mass spectrometric analysis revealed that turkey myoglobin is approximately 350 Da heavier in molecular mass than its beef and horse counterparts. Characterization of primary structure revealed that several smaller-sized AA in beef myoglobin were replaced with larger-sized ones in turkey (Joseph et al., 2011), for instance, substitutions at positions 5 (glycine with glutamine), 42 (lysine with arginine), 71 (alanine with glutamine), 148 (valine with glutamic acid), and 149 (leucine with phenyl alanine). These substitutions may contribute to increased thermostability and possibly provide a greater degree of protection to the heme protein against heat-induced denaturation.


Color of fresh meat is the major quality trait based on which consumers make their purchase decisions. A multitude of endogenous and exogenous factors contribute to color phenomena in meat from livestock and poultry, and the mechanistic interactions between myoglobin and other biomolecules in sarcoplasm govern the color stability. Innovative applications of proteomic tools revealed the fundamental basis of color biochemistry and indicated that meat color is species-specific, whereas within a species the trait is muscle-specific. The results of these investigations indicated the necessity to engineer species- and muscle-specific strategies to improve meat color.




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