<figure><img src="image_1.png" /></figure>

HAL
open science

# Proteomic investigations of beef tenderness

Brigitte Picard, Mohammed Gagaoua

► To cite this version:

Brigitte Picard, Mohammed Gagaoua. Proteomic investigations of beef tenderness. Proteomics in Food Science, Academic Press, 2017, 10.1016/B978-0-12-804007-2.00011-4. hal-01580641

HAL Id: hal-01580641

https://hal.science/hal-01580641v1

Submitted on 16 Oct 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Proteomic investigations of beef tenderness

Brigitte Picard¹, Mohammed Gagaoua¹,²

¹UMR1213 Herbivores, INRA, VetAgro Sup, Clermont université, université de Lyon,
63122 Saint-Genès-Champanelle, France

²INATAA, Université Frères Mentouri Constantine, Route de Ain El-Bey 25000,
Constantine, Algeria

Phone number/e-mail -Brigitte Picard: [brigitte.picard@clermont.inra.fr](mailto:brigitte.picard@clermont.inra.fr)

-Mohammed Gagaoua: [gmember2001@yahoo.fr](mailto:gmember2001@yahoo.fr)

**Abbreviated title:** Beef tenderness proteomics
**Abstract**

For the whole beef sector, from producers to consumers, the uncontrolled variability of beef tenderness is a major concern. For ten years, functional genomics and proteomics studies have been conducted to better understand the origin of this variability and to identify protein biomarkers that can be used to predict the tenderness of a piece of meat at slaughter or of a live animal. This chapter provides a comprehensive review on beef tenderness as provided by proteomics. Proteins representative of the major biological functions: heat shock proteins, energy metabolism, oxidative stress, structural proteins, proteolysis, transport, signaling and apoptosis, are described. The new knowledge thus produced are presented and discussed.

**Key words:** proteomics, beef, biomarkers, tenderness, proteins

**1. Introduction**

Tenderness, juiciness and flavor are important attributes of beef eating quality (Shackelford *et al*., 2001). Among them, tenderness is internationally the single, most important attribute affecting consumer's acceptability and their decision to purchase (Miller *et al*., 2001). The wide inconsistency of tenderness is the main reason for consumer's dissatisfaction leading to a reduction in beef consumption and has also been identified as a major problem for the beef industry (Brooks *et al*., 2000; Lorenzen *et al*., 1999). For decades researchers have focused on the mechanisms behind this important attribute. Muscle structure (connective tissue) and biochemical properties (proteolysis of myofibrillar and cytoskeletal proteins) are the primary factors influencing beef
tenderness variability (for review: Listrat *et al.*, 2016). However, the fine mechanisms controlling tenderness are still unclear (Ouali *et al.*, 2013). It is well documented that genetic and rearing factors, slaughter conditions and *post-mortem* ageing and processing, have significant effects on beef tenderization (Przybylski and Hopkins, 2015). All these factors are at the origin of the inconsistency of beef tenderness and explain the difficulty to control this quality attribute.

Tenderness can be evaluated by instrumental or sensory tools which are time-consuming and expensive (Platter *et al.*, 2003). Moreover, these methods can be applied only after the slaughter of the animal. There is still a need to find a universal method to evaluate the tenderness phenotype from paddock to plate. Among the omics tools developed over the last decade, proteomics appears to hold promise as a method to analyze protein modifications during *post-mortem* ageing and to reveal biomarkers of tenderness (for review: Picard *et al.*, 2013, 2015; Cassar-Malek and Picard, 2016). Meat ageing and processing involve many biochemical steps inducing changes in protein abundance and structure, consequently the use of proteomics appears to be relevant for understanding the *post-mortem* conversion of muscle into meat (Gagaoua *et al.*, 2015a,b). The importance of proteomics in the field of meat science is illustrated by a large number of papers and reviews concerning its applications to characterize the cellular and molecular mechanisms behind meat quality, skeletal muscle in the context of livestock production or biological traits and diseases in farm animals (Bendixen, 2005; Picard *et al.*, 2010; D'Allessando et Zolla, 2013; Franco *et al.*, 2015). The aim of this chapter is to summarize the main results obtained on beef tenderness investigations using proteomic tools. We will focus on the most relevant proteins related to beef tenderness identified through these approaches and discuss their biological roles on the process of muscle to meat conversion.
## 2. Proteomics in the field of meat science and investigations on beef tenderness

Most studies of skeletal proteome changes related to beef tenderness have been conducted by comparing extreme groups: tender *versus* tough for one muscle (Figure 1) using different proteomic methods (Table 1). Most of the studies have been performed on the *Longissimus thoracis* muscle, which is a valuable cut for steak. The innovations in bioinformatics with the development of databases, bioinformatic tools and web-services offer large possibilities of data-mining and interpretation of proteomic results. For example, the bioinformatic analysis of Guillemin *et al*. (2011) allowed the construction of a network of beef tenderness which revealed the main biological functions implicated and permitted the detection of differences between muscle types (*Longissimus thoracis*: LT vs. *Semitendinosus*: ST) identifying key proteins with central roles in the network such as *HspB1* coding for HSP27. This bioinformatic analysis proposed a number of proteins inside our dataset, but not revealed by the comparative proteomic analysis, as potential biomarkers of tenderness. For example, as depicted in Figure 2, Guillemin *et al*. (2011) showed in their network that three proteins: H2AFX, SUMO4 and TP53 were situated at crossroads between groups of proteins involved in tenderness, as metabolism, structure, and stress proteins. For example, H2AFX a histone involved in the cellular response to DNA damage, notably during oxidative stress, was confirmed by a proteomic study to be related with beef tenderness (D'Allessando and Zolla, 2013).

One limit of the electrophoretic separation used is the difficulty to extract proteins from extra-cellular matrices which are not solubilized by the buffer used. Hence, the proteins of connective tissue known to be important for meat tenderness are not considered in these studies. However,
one benefit of these methods is the possibility to study post-translational modifications (PTMs)
such as phosphorylation, glycosylation/glycation, oxidation and ubiquitination/sumoylation.
These PTMs are described to play major roles in the *post-mortem* process in meat science (Huang
*et al.*, 2011; D'Alessandro and Zolla, 2013). For example, phosphorylation is a reversible protein
modification that is known to affect the protein structure and activity of many enzymes *in vivo*,
and hence have a potential role in the tenderization period through regulation of the activities of
glycolytic enzymes. For example, myosin light chain (MyLC2) was doubly phosphorylated during
*rigor mortis* installation in bovine *Longissimus* muscle (Muroya *et al.*, 2007).

Comparative proteomic analyses using the approaches summarized in Table 1 allowed the identification of potential biomarkers of beef tenderness (Table 2) that belong to numerous biological pathways, namely the heat shock protein (HSP) family, glycolytic or oxidative metabolism, oxidative stress, muscle structure and contraction, and proteolysis (Figure 1 and Table 2). The strategy depicted in Figure 1 was to comprehensively identify proteins and evaluate and validate their relation with tenderness on large number of samples. The validated proteins are then used for tenderness prediction (Picard *et al.*, 2014). The final objective is to apply prediction equations in a paddock tool for the beef sector (Cassar-Malek and Picard, 2016).

3. Protein biomarkers of meat tenderness

The main biomarkers identified in relation to mechanical or sensory beef tenderness are
summarized in the Table 2. The analyses of these biomarkers and the biological functions in which
they are involved produced new insight about tenderness.
### 3.1. Heat shock proteins

In the majority of proteomic beef tenderness investigations, heat shock proteins (HSPs) have consistently been reported to differ between tender and tough beef samples and were also found to be correlated with instrumental and sensory tenderness (Bernard *et al.*, 2007; Morzel *et al.*, 2008; D'Alessandro *et al.*, 2012b; Bjarnadottir *et al.*, 2012; Carvalho *et al.*, 2014). HSPs are ubiquitous and evolutionarily conserved proteins classified into five subfamilies on the basis of their molecular weight (MW), e.g. HSP60, HSP70, HSP90 and HSP100 and the small HSPs (MW 12-43 kDa). In the field of meat science, Ouali *et al.* (2006) were the first who hypothesized the role of HSPs in meat tenderness due to their anti-apoptotic functions. Consequently, we speculated that HSPs are variably recruited to hinder the onset of apoptosis to prevent cells from damage, thus playing a great role in *post-mortem* muscle changes. As depicted in Figures 2 and 3, HSPs have essential roles in *post-mortem* skeletal muscle since they contribute to proper conformation of proteins and preservation of their biological functions. They act as molecular chaperones during protein assembly, protein folding and unfolding, and in the refolding of damaged proteins, thus maintaining cell homeostasis (Lomiwes *et al.*, 2014).

Skeletal muscle proteome studies have identified several HSPs that are changed in *post-mortem* bovine muscle (Pulford *et al.*, 2008; Jia *et al.*, 2009). Their increased abundance soon after slaughter may be due to their response to cell stress signals. Thus, we recently hypothesized that the up-regulation of the large HSP70 may be useful for maintaining muscle cell integrity and repairing denatured proteins (Picard *et al.*, 2014). In accordance, the abundance of large HSP70
proteins was found to be associated with meat tenderness (Jia *et al.*, 2009; D'Alessandro *et al.*, 2012a; Bjarnadóttir *et al.*, 2012; Carvalho *et al.*, 2014; Baldassini *et al.*, 2015). A negative relationship between tenderness and HSP40, a protein that helps to retard cell death in muscle tissue was reported (Bernard *et al.* (2007). Likewise, HSP27 was down-regulated in animals that gave lower tenderness values of *Longissimus* muscle (Morzel *et al.*, 2008). Furthermore, small HSPs are recruited to prevent structural damage and degradation of proteins due to apoptosis in muscle cells, and protect actin filaments and other cytoskeletal proteins from fragmentation caused by stress (Balan *et al.*, 2014). The modulation of these proteins during the *post-mortem* period suggests a change in the stabilization of the myofibril proteins (Jia *et al.*, 2007; Bjarnadóttir *et al.*, 2010). Considering that meat tenderness is dependent upon the degradation of myofibrillar proteins *post-mortem* (see section 3.4), small HSPs have therefore been implicated in the development of tenderness (Lomiweš *et al.*, 2014).

Previous studies by Pulford *et al.* (2008) and Kim *et al.* (2008) reported that the expression of HSP20 and αB-crystallin peaked at the early *post-mortem* of beef *Longissimus* muscle. On the other hand, it has been postulated that alterations in abundance of HSP20 may lead to decreased stability of the actin filament, which may be associated with increased tenderness (Zapata *et al.*, 2009). Protein levels of HSP70 were also found to be decreased in *Longissimus* muscle from Norwegian Red bulls 48 h *post-mortem*, as well as two spots corresponding to HSP27 (Bjarnadóttir *et al.*, 2010). These findings confirm that these chaperones are important in tenderness development, with HSP70 as negative markers, in contrast to HSP20 (Guillemin *et al.*, 2012). We have reported similar findings in a previous study showing significant higher levels of HSP27 and HSP20 in the more tender *Longissimus thoracis* in comparison to *Semitendinosus* muscle
(Guillemin *et al.*, 2011). Recently, an inverse relationship between tenderness and proteins from the small HSPs (HSP20, 27, and αB-crystallin) according to muscle type and breed has also been highlighted (Picard *et al.*, 2014). These proteins were found to be negatively related to tenderness in the *Semitendinosus* muscle and positively in the *Longissimus* muscle. It seems evident that the involvement of HSPs in beef tenderness is achieved through both their constitutive and inducible expression.

## 3.2. Energy metabolism

Several studies reporting *post-mortem* changes in beef (Jia *et al.*, 2006a,b; 2007; Laville *et al.*, 2009; Bjarnadóttir *et al.*, 2010) indicated a clear shift in energy metabolism *post-mortem* compared to the muscle of the living animal. Differentially abundant proteins between extreme groups of tenderness for *Longissimus thoracis* muscle, were glycolytic enzymes such as enolase, particularly enolase 3 (ENO3), malate dehydrogenase (MDH1), triosephosphate isomerase (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH A), pyruvate kinase (PKM) and phosphoglucomutase (PGM1) (Table 2). Relatively few proteins of oxidative metabolism were revealed: aldehyde dehydrogenase (ALDH2), creatine kinase M (CKM) and succinate dehydrogenase (SDH) (Table 2).

Enolase 1 (ENO1) and 3 (ENO3) are two isoforms catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. ENO1 is ubiquitous in cell cytoplasm, whereas ENO3 is mainly present in skeletal muscle. Their correlation to meat tenderness is isoform specific as ENO3 has been correlated with tenderness (Table 2). For
example, Jia *et al.*, (2006b) showed an increase of ENO3 early *post-mortem*. After longer ageing period (longer than 21 days), an increase of ENO3 fragments has been observed (Laville *et al.*, 2009), indicating the slowdown of the glycolytic process. Moreover, this protein has been reported to exhibit positive correlation with lightness (Gagaoua *et al.*, 2015c) and surface redness of beef (Joseph *et al.*, 2012).

An increase of PGM1 after 5 and 21 days of ageing has also been reported (Laville *et al.*, 2009). PGM1 catalyzes the production of 3-phosphoglycerate and ATP from 1,3-bisphosphoglycerate and is present as two isoforms: one with a strongly decreased concentration during the early *post-mortem* hours, while the other strongly increased only after longer ageing (Jia *et al.*, 2006a,b).

The enzyme PKM, involved in the catalysis of the transfer reaction of a phosphate group from phosphoenolpyruvate to ATP using a molecule of pyruvate, is found in the form of four isoenzymes among which two, PKM1 and PKM2, predominate in skeletal muscle. A relationship between gene polymorphism of PKM2 and glycogen content in the pig muscle has been reported with increased expression of PKM2 related to a more favorable evolution of glycolytic metabolism early *post-mortem* (Fontanesi *et al.*, 2003). Likewise, an increased amount of PKM2 correlates with decreased tenderness in Thai chicken meat (Mekchay *et al.*, 2010). One isoform of PKM2 which increases only after longer ageing was identified in beef (Table 3).
TPI, a glycolytic enzyme which catalyses the conversion of dihydroxyacetone phosphate to GAPDH, was reported to be related with WBSF (D'Alessandro et al., 2012b; Marino et al., 2014; Grabez et al., 2015). TPI was also proposed as a potential biomarker for intramuscular fat (Kim et al., 2008) and discoloration during *post-mortem* storage in beef (Wu et al., 2015).

The oxidative enzymes revealed to be related to tenderness are associated to mitochondria and
ATP regeneration (Ouali et al., 2013). In striated muscles, the protein CKM is associated with the
transfer of a phosphate group from phosphocreatine to ADP to generate ATP. An increase of
fragments of this protein during the ageing of meat has already been described (Jia et al., 2007;
Laville et al., 2009; Bjarnadóttir et al., 2010).

For the *Longissimus thoracis* muscle of young bulls from the main French beef breeds, a study by our group showed that several proteins representative of glycolytic metabolism were negatively related with tenderness (Chaze *et al.*, 2013). On the contrary, several proteins of oxidative metabolism were positively related with tenderness. This is in accordance with the above cited study of Morzel *et al*. (2008) showing that abundance of SDH was positively correlated with initial and overall tenderness in *Longissimus thoracis* muscle. We have recently validated these relationships in young bulls of French breeds (Picard *et al.*, 2014). However, we demonstrated that in Aberdeen Angus young bulls which are characterized by higher oxidative and less glycolytic metabolism comparatively to French beef breeds, these relationships were reversed. This is in accordance with the results of D'Alessandro *et al*. (2012a) showing a positive relation between proteins of glycolytic metabolism and tenderness in Chianina cattle (with oxidative muscles).
Laville *et al.* (2009) in Charolais young bulls also demonstrated that tender meat had higher abundance of mitochondrial membrane fragments in the early *post-mortem* period, suggesting increased degradation of the mitochondrial membrane in slow oxidative muscles. Mitochondria play a central role in the apoptotic process and a large set of pro-apoptotic proteins are translocated from these organelles to the cytoplasm and/or to the nucleus according to their biological function (Ouali *et al.*, 2013; Sierra and Olivan, 2013).

### 3.3. Oxidative stress

During muscle to meat conversion, muscle is subjected to various reactive oxygen species (ROS) which are able to target both the lipid and protein fractions (Brand, 2010). Among the protective scavenger agents revealed by proteomic beef tenderness studies, we can found peroxiredoxins (PRDX1, 2, 3 and 6), superoxide dismutase (SOD), DJ-1 (encoded by *PARK7*), aldehyde dehydrogenase (Jia *et al.*, 2009; Guillemin *et al.*, 2012; Polati *et al.*, 2012; Grabež *et al.*, 2015). Among them, SOD1 was positively correlated with WBSF in *Semitendinosus* muscle (Guillemin *et al.*, 2012). SOD is an antioxidant which allows a fast dismutation of O₂⁻ to O₂ and H₂O₂. In a recent study by Grabež and co-workers, superoxide dismutase (Cu-Zn) was more abundant in tough meat (Grabež *et al.*, 2015). The authors suggest that stronger oxidative stress may cause ROS accumulation in tough meat and reduce cell protection by Cu/Zn-SOD.

DJ-1, also known as *PARK7*, is another protein revealed by proteomic studies to be related to meat tenderness (Jia *et al.*, 2007; 2009; Polati *et al.*, 2012; Picard *et al.*, 2014; Gagaoua *et al.*, 2015b). This highly conserved protein has the ability to oxidize and translocate to mitochondria in response
to oxidative stress, thus serving a role as sensor and protector against toxicity from oxidative stress and limiting mitochondrial fragmentation (Thomas *et al.*, 2011). It was further reported to play an anti-apoptotic role by the involvement of its antioxidant activities. Furthermore, it has also been reported to increase in abundance in the bovine *Longissimus* muscle during the early tenderizing period (Polati *et al.*, 2012), after 10 h (Jia *et al.*, 2007) and after 5 and 21 days *post-mortem* (Laville *et al.*, 2009).

Among the PRDX family members reported to play an important role in the determination of beef tenderness and to maintain cell homeostasis, PRDX6 was identified as a potential marker of beef tenderness (Jia *et al.* (2009). PRDX6 is the only 1-cysteine peroxiredoxin that uses glutathione (GSH) rather than thioredoxin as the electron donor and is a bifunctional enzyme with both GSH peroxidase and acidic $Ca^{2+}$ independent phospholipase A2 (PLA2) activities (Fisher, 2011). This protein was found to be related with meat tenderness as well as other quality traits, such as pH decline and meat color (Gagaoua *et al.*, 2015c). Other PRDXs were also identified by proteomic studies (Table 2). PRDX1 is involved in regulating the cell redox state and plays an important role in the elimination of peroxide produced during metabolism. Polati *et al.* (2012) reported a decrease in the abundance of this protein after the early tenderizing period and a strong increase from early to late in the tenderization period. Further, thioredoxin and thioredoxin-dependent peroxide reductase (PRDX3), a mitochondrial precursor was more abundant in tender meat (Grabež *et al.*, 2015). Thus, thioredoxin/PRDX3 was suggested as a very efficient system concerning the regulation of ROS production in mitochondria (Jia *et al.*, 2007).
Finally, in addition to their role in glycolysis (Jia *et al.*, 2006b), identified mitochondrial ALDH are reported to protect cells from the oxidative stress through the oxidation of cytotoxic aldehyde derivates (Ohta *et al.*, 2004). Overall, the proteins identified to be involved in oxidative stress were proposed as negative biomarkers of tenderness mainly in glycolytic muscle types (Guillemin *et al.*, 2012). However, it is presently difficult to get conclusions about their real role in *post-mortem* muscle and thus, more investigations are needed in order to clarify the exact relationship with ultimate meat tenderness.

### 3.4. Structural proteins

During *post-mortem* tenderization there are major changes in the myofibrillar structure and thanks to proteomics, their study and identification were possible (Lametsch *et al.*, 2003). Among the structural proteins identified by proteomic investigation that were sown to be related to beef tenderness, we found α-actin (Lametsch *et al.*, 2003; Hwang *et al.*, 2005; Morzel *et al.*, 2008; Zapata *et al.*, 2009; Bjarnadóttir *et al.*, 2012; Polati *et al.*, 2012). This protein was more abundant in the tender group in the *Longissimus* muscle of young bull from three French breeds (Chaze *et al.*, 2013). The authors also identified the structural protein CapZβ with a higher abundance in the tender group. A study by Laville *et al.* (2009) identified an actin fragment of 31 kDa to be upregulated in the tender meat of Charolais young bulls compared to tougher Charolais meat. Ouali *et al.* (2013) suggested that α-actin, a major constituent of contractile apparatus in muscle tissue, constitutes a good marker of *post-mortem* apoptosis. It was found to be modulated during maturation of bovine muscle (Bjarnadóttir *et al.*, 2010). Full-length α-actin was also observed with decreased abundance in the tender group by iTRAQ and 2-DE (Kim *et al.*, 2008; Laville *et al.*,
2009; Bjarnadóttir *et al.*, 2015). Alpha-actin has also been reported to be present in less abundance in bovine muscle samples taken 36 h *post-mortem* and to correlate with WBSF at 72 h *post-mortem* (*Zapata et al.*, 2009).

Chaze *et al.* (2013) demonstrated a lower abundance of several spots of TnnTfast in the tender groups of French beef breeds. Moreover, it has also been shown that in bovine *Longissimus* (Jia *et al.*, 2006b, 2007) and in pig muscles (Lametsch *et al.*, 2002) intact TnnT decreases during the *post-mortem* period, while its fragments increase.

Myosin heavy chain (MyHC) fragmentation of bovine *Longissimus* muscle was found to correlate with tenderness at 7 days (Sawdy *et al.*, 2004). It was shown that heavy (MyH1) and light (MyL1, MyL2, and MyL6B) myosin II chains have altered abundance and notably most fragmented chains accumulate during tenderization. Two myosin light chain MLC1 and 2 were reported to be closely related to tenderness of meat by several authors (Zapata *et al.*, 2009; Jia *et al.*, 2009). Bjarnadóttir *et al.* (2012) reported that MLC1 was more abundant in muscle biopsies from tender meat, while MLC2 were present in less abundance in tender meat. The correlation with tenderness of the two MyHC-I (MyH7) and MyHC-IIx (MyH1) was specific of muscle contraction type. Correlation with tenderness of the fast-twitch MyHC was positive in fast-twitch muscles, and negative in slow-twitch muscles (Picard *et al.*, 2014).
Anderson *et al.* (2012) in beef *Adductor* (tough muscle) and *Longissimus* (moderately tender muscle) revealed that proteins such as actin, MyH1 fragment, myomesin-2, and α-actinin-3 undergo muscle specific changes during aging.

### 3.5. Proteolysis

The enzymatic machinery that underlies muscle to meat conversion involves synergistic activities of numerous endogenous proteolytic enzymes. There are several proteolytic systems present in muscle, which could participate in *post-mortem* proteolysis, including the calpain, cathepsin, proteasome systems and more recently caspases (Sentandreu *et al.*, 2002; Ouali *et al.*, 2006; 2013). These proteolytic systems has been extensively reviewed (Sentandreu *et al.*, 2002; Huff Lonergan *et al.*, 2010), thus are not discussed in this chapter but only the input of proteomics is presented.

In a proteomic-based approach, putative partners of calpain enzymes were revealed (Brulé *et al.*, 2010). Calpains are members of a large family of 14 related calcium-dependent cysteine proteases. The isoforms μ-calpain and m-calpain are respectively two typical and ubiquitous isoforms consisting of a distinct large catalytic subunit, CAPN1 or CAPN2, encoded by *capn1* and *capn2* genes respectively (Goll *et al.*, 2003). Both enzymes were found to interact with several proteins belonging to numerous biological pathways, namely Ca²⁺ homeostasis, structure, glucose metabolism, heat stress, mitochondria and apoptosis. The association between Ca²⁺-regulating proteins and Ca²⁺-dependent calpains suggest a complex nature of the mechanisms governing *post-mortem* proteolysis and thus, meat tenderness. For example, ryanodine receptor (Brulé *et al.*, 2010) and oxidized HSP70-1A/B (Gagaoua *et al.*, 2015c) were shown to be potential substrates for
calpains. Furthermore, the use of proteomic tools revealed that μ-calpain undergoes oxidation by
forming an intermolecular disulfide bond and that the oxidation results in the loss of proteolytic
activity. This finding highlighted the adverse influence of protein oxidation on meat tenderness.
Moreover, changes in the proteome of purified myofibrils caused by a four-day *in vitro* incubation
with CAPN1 under simulated *post-mortem* conditions, was reported (Lametsch *et al.*, 2004).
Recently, with analyses of bovine muscle proteome using protein-protein interactions and
correlation networks, additional protein partners (potential interacting proteins) rather than
calpastatin (a specific inhibitor of calpains) were revealed to be associated with these enzymes
(Guillemin *et al.*, 2011; Gagaoua *et al.*, 2015b).

Furthermore, both proteases were used in an immunological tool as biomarkers to predict beef
tenderness (Guillemin *et al.*, 2011; 2012; Picard *et al.*, 2014). Guillemin *et al.* (2011) showed that
the proteolysis contribution to tenderness is different between *Semitendinosus* and *Longissimus*
muscles. In *Semitendinosus*, they found a positive correlation with tenderness for CAPN1, in
contrast to the negative correlation for CAPN2. As CAPN1 is known to play a major role in
tenderness, they supposed that in *Semitendinosus* the role of CAPN2 is less important. The authors
stressed forward that this inverse relation comparatively to *Longissimus* is in coherence with the
inverse correlation between contractile and metabolic proteins with tenderness in the two muscles,
already observed by Picard *et al*. (2014).

**3.6. Transport, signaling and apoptosis**
In addition to the above discussed proteins, numerous other biological pathways such as transport, signaling and apoptosis are reported. Several of the proteins presented above are also related to apoptosis. The hypothesis of the involvement of apoptotic programmed cell death by muscle cells was first proposed by Ouali *et al.* (2006) as a possible explanation of the high variability in meat tenderness. Later on, numerous studies showed close connections between the apoptopic pathway and meat tenderization (Kemp and Parr, 2012; Ouali *et al.*, 2013; Sierra and Olivan, 2013; Gagaoua *et al.*, 2012; 2015a).

Among the identified proteins we found the annexins (Table 2), which are members of a large structurally-related and Ca²⁺ sensitive protein family. Expressed in all eukaryotic cells, they participate in a variety of cellular processes including apoptosis and intracellular signaling (Monastyrskaya *et al.*, 2009). Bjarnadottir *et al.* (2012) observed lower annexin A6 levels in tender beef meat. In *post-mortem* muscle, annexin A6 may act as a brake to apoptosis through inhibition of dynamin related protein 1 and subsequent intensification of cytochrome C release occurring soon after animal death (Ouali *et al.*, 2013). Bjarnadottir *et al.* (2012) suggested that the lower abundance of annexin A6 in the tender samples may lead to an accelerated release of Ca²⁺ and thus affect the activity of calpains. Furthermore, a change in the concentration of annexin A1 (also known as lipocortin-1) probably related to apoptosis development, has also been reported to change in *post-mortem* muscle (Zhao *et al.*, 2010).

Galectin 1, a β-galactose-binding lectin involved in apoptotic process, was proposed to be linked with beef tenderness (Zapata *et al.*, 2009; Bjarnadottir *et al.*, 2012). Both studies suggested that a
lower expression of galectin 1 is associated with greater tenderness. However, these somewhat preliminary results warrant further investigations since proteomic studies have yet to confirm these findings.

Numerous *post-mortem* events are controlled by muscle Ca²⁺ homeostasis (Ouali *et al.*, 2013) and it is well documented that proteins involved in Ca²⁺ regulation are important for meat quality (Picard *et al.*, 2010). Parvalbumin was found more abundant in tender muscles in Charolais and Limousin cattle (Bouley *et al.*, 2004b). Parvalbumin has a strong affinity for Ca²⁺ ions and has been reported to participate in their transfer between cytoplasm and sarcoplasmic reticulum (Berchtold *et al.*, 2000) especially in fast fibers. Inositol 1,4,5-triphosphate receptor type 1 (IP3R1), is another protein involved in Ca²⁺ channel activity that was reported by Kim *et al*. (2008) to be higher in animal groups with superior tenderness.

**4. Protein biomarkers and their role in determining beef tenderness**

Bioinformatics and statistical analyses of the identified proteins have elucidated the biological mechanisms behind beef tenderness development (Figure 4). Knowledge of the biological mechanisms allowed the construction of a correlation network between several biomarkers of tenderness (Gagaoua *et al.*, 2015). This revealed some robust correlations as they were observed in three continental breeds and two muscles studied. These correlations allowed a better understanding of the underlying mechanisms. For example, the protein DJ-1 was found to be positively correlated to small HSP (HSP20, HSP27) and to glycolytic enzymes such as Eno3, LDH-B and MDH1. These correlations originated through the oxidative stress as DJ-1 was
reported to play an anti-apoptotic role by the involvement of its anti-oxidant activities (Fan *et al.*, 2008). These authors reported that deficiency in DJ-1 induced an increase of apoptosis through a decrease of *Bax*, a pro-apoptotic protein regulator, expression and an inhibition of caspase activation, thus affecting muscle structures and meat tenderness development. As such, we proposed that the *post-mortem* increase of ROS production would increase anti-oxidant and sHSP proteins expression (Figure 4).

Another robust correlation was found between μ-calpain and PRDX6 (Gagaoua *et al.*, 2015). It was hypothesized that the correlation could be explained by the PLA2 group of PRDX6 being able to hydrolyze phosphatidylcholine. They proposed that at the first stage of apoptosis *post-mortem*, the inversion of membrane polarity would induce modifications in the fluidity of the membrane increasing the permeability to ions such as Ca²⁺ and increasing the μ-calpain activity. The activity of μ-calpain was also found to be correlated with several members of HSP70 family in LT muscle, but not in ST muscle. HSP70 expression is induced by hypoxic and ischemic conditions and by ROS production (Wang *et al.*, 2012). They are involved in apoptotic pathways through the sequestration of *Bcl-2* pro-apoptotic factor. Under oxidative stress conditions, HSP70 are also reported to collaborate with HSP40, a biomarker of beef tenderness and play an anti-apoptotic action (Bernard *et al.*, 2007). According to Gotoh *et al.* (2004), the anti-apoptotic actions may begin with the formation of a complex with active caspases and slowdown the process of cellular death which will constitute an obstacle to good meat ageing and consequently to meat tenderness. Further, HSPs may also sequester cytochrome C after its release from the mitochondria (Bruey *et al.*, 2000). Finally, the ROS accumulation can trigger both autophagy and apoptosis, but their precise roles are not known, and their establishment is strongly variable. Further, oxidative stress
and mitochondrial metabolism seem to play major role in initiating a cascade of reactions affecting
the determinism of tenderness (Figure 4).

**5. Conclusion and future prospects**

Proteomic studies applied to beef tenderness over the last 15 years has led to substantial increase
in knowledge in this field confirming the important role of energy metabolism and proteolysis in
muscle to meat conversion. Furthermore, proteomics studies have implicated biological functions
such as apoptosis, oxidative stress and autophagy in tenderness. The cumulative findings give
researchers a better understanding of the *post-mortem* modifications and the impact on meat
tenderness development. Muscle type and breed effects have been elucidated identifying biological
pathways that are muscle-type specific and others independent of the muscle-type (Picard *et al.*,
2014; Gagaoua *et al.*, 2015c). Proteins linked to specific biological functions have been proposed
as biomarkers which could see application in the future for routine prediction of tenderness. The
development of phenotyping tools, making use of these biomarkers, may in time be used by
livestock farmers, breeders and industrials of the beef sector either on carcasses or on live animals.
The recent advancements in proteomic technologies and availability of bioinformatic tools
(Kaspric *et al.*, 2015) will enable further progress in biomarker discovery and quantification.
Moreover, the proteomic strategy applied to beef tenderness starts to be applied to other meat traits,
such as color (Joseph *et al.*, 2012), pH drop (Damon *et al.*, 2013; Gagaoua *et al.*, 2015c; Di Luca
*et al.*, 2016), water holding capacity (Di Luca *et al.*, 2016; Gagaoua *et al.*, 2016), juiciness and
flavor (Cassar-Malek and Picard, 2016), and adiposity (Damon *et al.*, 2006; Kim *et al.*, 2008;
Mao *et al.*, 2016).
<figure><img src="image_3.png" /></figure>
<table><caption>**Table 1.** A summary of gel-based and gel-free techniques used for quantitative beef proteomic studies.</caption><thead><tr><td>Approach</td><td>Objective</td><td>References</td></tr></thead><tbody><tr><td rowspan="2">1-DE</td><td>Relationship between myofibrillar 1-D protein patterns of <i>Longissimus</i> muscle with tenderness</td><td>(Sawdy et al. 2004)</td></tr><tr><td>Beef tenderness prediction using separated myofibrillar 1-D protein bands</td><td>(Zapata et al. 2009)</td></tr><tr><td rowspan="3">DIGE</td><td>Assess the changes in the myosin light chains of <i>Longissimus</i> myofibril proteins during <i>p-m</i> aging</td><td>(Muroya et al. 2007)</td></tr><tr><td>Identification of protein markers of meat tenderness in <i>Longissimus</i> muscle</td><td>(Jia et al. 2009)<br>(Anderson et al. 2012)</td></tr><tr><td>Explore the basis of meat variation in tenderness by studying the changes in protein abundances</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td>iTRAQ</td><td>Identification of the differentially expressed proteins involved in intramuscular fat deposition</td><td>(Mao et al. 2016)</td></tr><tr><td rowspan="2">MALDI-TOF</td><td>Mapping of bovine <i>Semitendinosus</i> muscle of Charolais cattle</td><td>(Bouley et al. 2004a)</td></tr><tr><td>Changes of meat quality in <i>Longissimus</i> muscle following water bath and ohmic cooking</td><td>(Tian et al. 2016)</td></tr><tr><td rowspan="2">LC-ESI-MS</td><td>Identification of early predictors of meat eating qualities of the Blonde d'Aquitaine bulls</td><td>(Morzel et al. 2008)</td></tr><tr><td>Separation and identification of the potential proteins explaining the variability of beef tenderness</td><td>(Baldassini et al. 2015)</td></tr><tr><td rowspan="3">LC-MS/MS</td><td>Relationship between changes in <i>p-m</i> degradation of myofibrillar proteins and tenderness development</td><td>(Marino et al. 2015)</td></tr><tr><td>Phosphorylation of sarcoplasmic and myofibrillar proteins in response to <i>p-m</i> electrical stimulation</td><td>(Li et al. 2015)</td></tr><tr><td>Explore the proteome changes in the <i>Longissimus</i> muscle in response to pre-slaughter stress</td><td>(Franco et al. 2015)</td></tr><tr><td rowspan="2">nLC-MS/MS</td><td>Explore the effect of high pressure on skeletal muscle proteome and the induced changes</td><td>(Marcos and Mullen 2014)</td></tr><tr><td>Occurrence of low MW peptides in the <i>Pectoralis</i> muscle during ageing and cooking</td><td>(Bauchart et al. 2006)</td></tr><tr><td>LESA-MS</td><td>Analysis of skeletal muscle proteins derived from a mixture of standard proteins and raw meat</td><td>(Montowska et al. 2014)</td></tr></tbody></table>

1-DE, one dimensional electrophoresis; DIGE, Difference Gel Electrophoresis; iTRAQ, isobaric Tag for Relative and Absolute Quantification; MALDI-TOF, Matrix Assisted Laser Desorption/Ionization-time-of-flight; LC-ESI-MS, Liquid chromatography-electrospray ionization-tandem mass spectrometry; nLC-MS/MS, nanoliquid chromatography-tandem mass spectrometry; LESA-MS, Liquid Extraction Surface Analysis Mass Spectrometry
<table><caption>**Table 2.** Selected protein markers identified by proteomics approach associated with the variability of beef tenderness.</caption><thead><tr><th>Protein biomarkers (gene name)</th><th>Tenderness trait <sup>1</sup></th><th>Direction <sup>2</sup></th><th>References</th></tr></thead><tbody><tr><td colspan="4"><strong>Heat shock proteins (HSP)</strong></td></tr><tr><td rowspan="3">HSP20 (HspB6)</td><td rowspan="3">WBSF</td><td>+</td><td>(Zapata et al. 2009)</td></tr><tr><td>+</td><td>(Guillemin et al. 2011)</td></tr><tr><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td rowspan="3">HSP27 (HspB1)</td><td>WBSF</td><td>-</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>WBSF</td><td>+/-</td><td>(D'Alessandro et al. 2012a)</td></tr><tr><td>WBSF</td><td>+</td><td>(Kim et al. 2008)</td></tr><tr><td rowspan="4">αB-crystallin (Cryab)</td><td>WBSF</td><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>WBSF</td><td>-</td><td>(Carvalho et al. 2014)</td></tr><tr><td>WBSF</td><td>-/-**</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>Tenderness (S)</td><td>+/-</td><td>(Morzel et al. 2008)</td></tr><tr><td rowspan="4">HSP40</td><td>WBSF</td><td>+</td><td>(Zapata et al. 2009)</td></tr><tr><td>WBSF</td><td>-</td><td>(Guillemin et al. 2011)</td></tr><tr><td>WBSF</td><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>Tenderness (S)</td><td>**</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>HSP71 (HspA8)</td><td>WBSF</td><td>-</td><td>(Bernard et al. 2007)</td></tr><tr><td rowspan="3">HSP70-1A (HspA1A)</td><td rowspan="3">WBSF</td><td>+</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>+</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td>-</td><td>(Carvalho et al. 2014)</td></tr><tr><td rowspan="2">HSP70-1B (HspA1B)</td><td rowspan="2">WBSF</td><td>+</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>-</td><td>(Baldassini et al. 2015)</td></tr><tr><td rowspan="3">HSPp70-GRP75 (HSPA9)</td><td rowspan="3">WBSF</td><td>+</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>-</td><td>(Jia et al. 2009)</td></tr><tr><td>-</td><td>(Guillemin et al. 2011)</td></tr><tr><td colspan="4"><strong>Energy metabolism</strong></td></tr><tr><td colspan="4"><strong>A) Glycolytic metabolism</strong></td></tr><tr><td rowspan="2">Enolase 1 (ENO1)</td><td rowspan="2">WBSF</td><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>-</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td rowspan="4">Enolase 3 (ENO3)</td><td rowspan="4">WBSF</td><td>-</td><td>(Bjarnadottir et al. 2012).</td></tr><tr><td>+</td><td>(Guillemin et al. 2011)</td></tr><tr><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>+/-</td><td>(Marino et al. 2014)</td></tr><tr><td>Malate dehydrogenase 1 (MDH1)</td><td>WBSF</td><td>-</td><td>(Jia et al. 2009)</td></tr><tr><td rowspan="2">Triosephosphate isomerase 1 (TPII)</td><td rowspan="2">WBSF</td><td>-</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td>-</td><td>(Grabez et al. 2015)</td></tr><tr><td rowspan="4">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</td><td rowspan="4">WBSF</td><td>**</td><td>(Polati et al. 2012)</td></tr><tr><td>+</td><td>(Laville et al. 2009)</td></tr><tr><td>-</td><td>(D'Alessandro et al. 2012a)</td></tr><tr><td>**</td><td>(Marino et al. 2014)</td></tr><tr><td rowspan="2">Lactate dehydrogenase A (LDHA)</td><td rowspan="2">WBSF</td><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>*/+</td><td>(Baldassini et al. 2015)</td></tr><tr><td rowspan="2">Pyruvate kinase (PKM)</td><td rowspan="2">WBSF</td><td>+</td><td>(Baldassini et al. 2015)</td></tr><tr><td>-</td><td>(Laville et al. 2009)</td></tr><tr><td>Phosphoglucomutase 1 (PGM1)</td><td>WBSF</td><td>-</td><td>(D'Alessandro et al. 2012b)</td></tr><tr><td colspan="4"><strong>B) Oxidative metabolism</strong></td></tr><tr><td rowspan="2">Aldehyde dehydrogenase (ALDH2)</td><td rowspan="2">WBSF</td><td>-</td><td>(Grabez et al. 2015)</td></tr><tr><td>*/**</td><td>(Jia et al. 2007)</td></tr><tr><td>Creatine kinase M type (CKM)</td><td>WBSF</td><td>-</td><td>(Zapata et al. 2009)</td></tr></tbody></table>
<table><caption>**Table 2. (Continued)**</caption><tr><td></td><td></td><td>-</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td></td><td></td><td>*/**</td><td>(Polati et al. 2012)</td></tr><tr><td></td><td></td><td>-</td><td>(Laville et al. 2009)</td></tr><tr><td></td><td></td><td>-</td><td>(D'Alessandro et al. 2012a)</td></tr><tr><td>Succinate dehydrogenase (SDH)</td><td>Tenderness (S)</td><td>+</td><td>(Morzel et al. 2008)</td></tr><tr><td>Pyruvate dehydrogenase (PDHB)</td><td>WBSF</td><td>-</td><td>(Grabez et al. 2015)</td></tr></table>

<table><thead><tr><th>Protein biomarkers (gene name)</th><th>Tenderness trait <sup>1</sup></th><th>Direction <sup>2</sup></th><th>References</th></tr></thead><tbody><tr><td colspan="4"><strong>Oxidative stress</strong></td></tr><tr><td>DJ-1 (PARK7)</td><td>WBSF</td><td>+</td><td>(Jia et al. 2009)</td></tr><tr><td></td><td></td><td>*</td><td>(Jia et al. 2007)</td></tr><tr><td></td><td></td><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>Peroxiredoxin 1 (PRDX1)</td><td>WBSF</td><td>*</td><td>(Polati et al. 2012)</td></tr><tr><td>Peroxiredoxin 2 (PRDX2)</td><td>WBSF</td><td>*</td><td>(Jia et al. 2007)</td></tr><tr><td>Peroxiredoxin 3 (PRDX3)</td><td>WBSF</td><td>*</td><td>(Jia et al. 2007)</td></tr><tr><td></td><td></td><td>-</td><td>(Jia et al. 2009)</td></tr><tr><td>Peroxiredoxin 6 (PRDX6)</td><td>WBSF</td><td>+</td><td>(Guillemin et al. 2011)</td></tr><tr><td></td><td></td><td>*</td><td>(Marino et al. 2014)</td></tr><tr><td>Superoxide dismutase (SOD)</td><td>WBSF</td><td>*</td><td>(Marino et al. 2014)</td></tr><tr><td></td><td></td><td>-</td><td>(Grabez et al. 2015)</td></tr><tr><td>Carbonic anhydrase (CA3)</td><td>WBSF</td><td>**</td><td>(Polati et al. 2012)</td></tr><tr><td colspan="4"><strong>Structure</strong></td></tr><tr><td>Myosin light chain 1 (MLC1)</td><td>WBSF</td><td>+</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td></td><td></td><td>-</td><td>(Jia et al. 2009)</td></tr><tr><td></td><td></td><td>+/-</td><td>(Polati et al. 2012)</td></tr><tr><td>Myosin light chain 2 (MLC2)</td><td>WBSF</td><td>+</td><td>(Zapata et al. 2009)</td></tr><tr><td></td><td></td><td>-</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td></td><td></td><td>+/-</td><td>(Polati et al. 2012)</td></tr><tr><td>Troponin C</td><td>WBSF</td><td>+</td><td>(Zapata et al. 2009)</td></tr><tr><td>Troponin T</td><td>WBSF</td><td>+</td><td>(Polati et al. 2012)</td></tr><tr><td></td><td></td><td>-</td><td>(Zapata et al. 2009)</td></tr><tr><td>Myosin heavy chain 1 (MHC1)</td><td>WBSF</td><td>-</td><td>(Guillemin et al. 2011)</td></tr><tr><td></td><td></td><td>+/-</td><td>(Polati et al. 2012)</td></tr><tr><td>Myosin heavy chain 2 (MHC2)</td><td>WBSF</td><td>-</td><td>(Zapata et al. 2009)</td></tr><tr><td>Myosin heavy chain 7 (MHC7)</td><td>WBSF</td><td>-</td><td>(Zapata et al. 2009)</td></tr><tr><td></td><td></td><td>+</td><td>(Guillemin et al. 2011)</td></tr><tr><td>Desmin</td><td>WBSF</td><td>-</td><td>(Zapata et al. 2009)</td></tr><tr><td></td><td></td><td>-</td><td>(Guillemin et al. 2011)</td></tr><tr><td>Tubulin β</td><td>WBSF</td><td>-</td><td>(Zapata et al. 2009)</td></tr><tr><td>CapZ-β</td><td>WBSF</td><td>*/**</td><td>(Bjarnadottir et al. 2010)</td></tr><tr><td></td><td>WBSF</td><td>-</td><td>(Zapata et al. 2009)</td></tr><tr><td></td><td>WBSF</td><td>+</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td>α-actin</td><td>WBSF</td><td>+/-</td><td>(Polati et al. 2012)</td></tr><tr><td></td><td>Tenderness (S)</td><td>+</td><td>(Morzel et al. 2008)</td></tr><tr><td>Myosin binding protein-H (MBP-H)</td><td>WBSF</td><td>+</td><td>(Guillemin et al. 2011)</td></tr><tr><td></td><td>Tenderness (S)</td><td>+</td><td>(Morzel et al. 2008)</td></tr><tr><td colspan="4"><strong>Proteolysis</strong></td></tr><tr><td>μ-calpain</td><td>Tenderness (S)</td><td>-</td><td>(Guillemin et al. 2011)</td></tr><tr><td></td><td>WBSF</td><td>+/-</td><td>(Picard et al. 2014)</td></tr><tr><td>m-calpain</td><td>Tenderness (S)</td><td>-</td><td>(Guillemin et al. 2011)</td></tr><tr><td colspan="4"><strong>Transport and signaling</strong></td></tr><tr><td>Serum albumin</td><td>WBSF</td><td>-</td><td>(Laville et al. 2009)</td></tr></tbody></table>
<table><tr><td>Serotransferrin</td><td>WBSF</td><td>+</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td>Parvalbumin</td><td>Tenderness (S)</td><td>+</td><td>(Bouley et al. 2004b)</td></tr><tr><td>Myoglobin</td><td>WBSF</td><td>+/-</td><td>(Polati et al. 2012)</td></tr><tr><td>Fatty acid-binding protein (FABP)</td><td>WBSF</td><td>+/-</td><td>(Polati et al. 2012)</td></tr><tr><td colspan="4"><strong>Apoptosis <sup>3</sup></strong></td></tr><tr><td>Annexin A6</td><td>WBSF</td><td>+</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td>Galectin-1</td><td>WBSF</td><td>+</td><td>(Bjarnadottir et al. 2012)</td></tr><tr><td>Inositol 1,4,5-triphosphate receptor type 1 (IP3R1)</td><td>WBSF</td><td>+</td><td>(Kim et al. 2008)</td></tr></table>

<footnote n="1">¹ Tenderness was evaluated by instrumental Warner-Bratzler Shear Force (WBSF) or by a sensory panel (S).</footnote>

<footnote n="2">² (+) positively related; (-) negatively related; (+/-) positively and/or negatively related depending on the protein isoform; (*) increase of the biomarker was reported; (**) decrease of the biomarker was reported.</footnote>

<footnote n="3">³ Among the HSP, transport and signaling, and oxidative stress proteins, many of these proteins are also involved in the apoptotic pathway.</footnote>
**Figure captions**

**Figure 1.** A workflow of the proteomic strategy adopted for the discovery of protein biomarkers of beef tenderness.

**Figure 2.** Functional network of tenderness in *post-mortem* muscle (adapted from Guillemin *et al*., 2011) Functional interactions between proteins are symbolized with arrow, with a brief description. Heavy arrows indicate cellular functions assured by the proteins. HSF: Heat shock factor.

**Figure 3.** Potential roles of heat shock proteins (HSPs) in *post-mortem* muscle to meat conversion.

**Figure 4.** Postulated biological mechanisms of *post-mortem* muscle to meat conversion involving numerous biological pathways proposed thanks to proteomics.

**References**
Anderson M.J., Lonergan S.M. & Huff-Lonergan E. (2012) Myosin light chain 1 release from myofibrillar fraction during postmortem aging is a potential indicator of proteolysis and tenderness of beef. *Meat Sci* 90, 345-351.

Anderson N.L. & Anderson N.G. (1998) Proteome and proteomics: New technologies, new concepts, and new words. *Electrophoresis* 19, 1853-1861.

Balan P., Brad Kim Y.H. & Blijenburg R. (2014) Small heat shock protein degradation could be an indicator of the extent of myofibrillar protein degradation. *Meat Sci*, 97, 220-222.

Baldassini W.A., Braga C.P., Chardulo L.A., Vasconcelos Silva J.A., 2nd, Malheiros J.M., de Albuquerque L.G., Fernandes T.T. & Padilha Pde M. (2015) Bioanalytical methods for the metalloproteomics study of bovine longissimus thoracis muscle tissue with different grades of meat tenderness in the Nellore breed (Bos indicus). *Food Chem* 169, 65-72.

Bauchart C., Remond D., Chambon C., Patureau Mirand P., Savary-Auzeloux I., Reynes C. & Morzel M. (2006) Small peptides (<5kDa) found in ready-to-eat beef meat. *Meat Sci* 74, 658-666.

Bendixen E. (2005) The use of proteomics in meat science. *Meat Sci* 71, 138-149.

Berchtold M.W., Brinkmeier H. & Müntener M. (2000) Calcium Ion in Skeletal Muscle: Its Crucial Role for Muscle Function, Plasticity, and Disease. *Physiological Reviews* 80, 1215-1265.

Bernard C., Cassar-Malek I., Le Cunff M., Dubroeucq H., Renand G. & Hocquette J.F. (2007) New indicators of beef sensory quality revealed by expression of specific genes. *J Agric Food Chem* 55, 5229-5237.

Bjarnadottir S.G., Hollung K., Faergestad E.M. & Veiseth-Kent E. (2010) Proteome changes in bovine longissimus thoracis muscle during the first 48 h postmortem: shifts in energy status and myofibrillar stability. *J Agric Food Chem* 58, 7408-7414.
Bjarnadottir S.G., Hollung K., Hoy M., Bendixen E., Codrea M.C. & Veiseth-Kent E. (2012)
Changes in protein abundance between tender and tough meat from bovine longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis. *J Anim Sci* 90, 2035-2043.

Bouley J., Chambon C. & Picard B. (2004a) Mapping of bovine skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry. *Proteomics* 4, 1811-1824.

Bouley J., Meunier B., Culioli J. & Picard B. (2004b) Analyse protéomique du muscle de Bovin appliquée à la recherche de marqueurs de la tendreté de la viande. *Renc. Rech. Rum* 11, 87-89.

Brand M.D. (2010) The sites and topology of mitochondrial superoxide production. *Experimental Gerontology* 45, 466-472.

Brooks J.C., Belew J.B., Griffin D.B., Gwartney B.L., Hale D.S., Henning W.R., Johnson D.D., Morgan J.B., Parrish F.C., Reagan J.O. & Savell J.W. (2000) National Beef Tenderness Survey-1998. *J Anim Scie* 78, 1852-1860.

Bruey J.-M., Ducasse C., Bonniaud P., Ravagnan L., Susin S.A., Diaz-Latoud C., Gurbuxani S., Arrigo A.-P., Kroemer G., Solary E. & Garrido C. (2000) Hsp27 negatively regulates cell death by interacting with cytochrome c. *Nat Cell Biol* 2, 645-652.

Brulé C., Dargelos E., Diallo R., Listrat A., Béchet D., Cottin P. & Poussard S. (2010) Proteomic study of calpain interacting proteins during skeletal muscle aging. *Biochimie* 92, 1923-1933.

Carvalho M.E., Gasparin G., Poleti M.D., Rosa A.F., Balieiro J.C.C., Labate C.A., Nassu R.T., Tullio R.R., Regitano L.C.d.A., Mourão G.B. & Coutinho L.L. (2014) Heat shock and structural proteins associated with meat tenderness in Nellore beef cattle, a Bos indicus breed. *Meat Science* 96, 1318-1324.
Cassar-Malek I. & Picard B. (2016) Expression Marker-Based Strategy to Improve Beef Quality.
*Scientific World Journal* 2016, 2185323.

Chaze T., Hocquette J.-F., Meunier B., Renand G., Jurie C., Chambon C., Journaux L., Rousset S., Denoyelle C., Lepetit J. & Picard B. (2013) Biological Markers for Meat Tenderness of the Three Main French Beef Breeds Using 2-DE and MS Approach. In: *Proteomics in Foods* (eds. by Toldrá F & Nollet LML), pp. 127-146. Springer US.

D'Alessandro A., Marrocco C., Rinalducci S., Mirasole C., Failla S. & Zolla L. (2012a) Chianina beef tenderness investigated through integrated Omics. *J Proteomics* 75, 4381-4398.

D'Alessandro A., Rinalducci S., Marrocco C., Zolla V., Napolitano F. & Zolla L. (2012b) Love me tender: an Omics window on the bovine meat tenderness network. *J Proteomics* 75, 4360-4380.

D'Alessandro A. & Zolla L. (2013) Meat science: From proteomics to integrated omics towards system biology. *J Proteomics* 78, 558-577.

Damon M., Denieul K., Vincent A., Bonhomme N., Wyszynska-Koko J. & Lebret B. (2013) Associations between muscle gene expression pattern and technological and sensory meat traits highlight new biomarkers for pork quality assessment. *Meat Sci* 95, 744-754.

Damon M., Louveau I., Lefaucheur L., Lebret B., Vincent A., Leroy P., Sanchez M.P., Herpin P. & Gondret F. (2006) Number of intramuscular adipocytes and fatty acid binding protein-4 content are significant indicators of intramuscular fat level in crossbred Large White × Duroc pigs. *Journal of Animal Science* 84, 1083-1092.

Di Luca A., Hamill R.M., Mullen A.M., Slavov N. & Elia G. (2016) Comparative Proteomic Profiling of Divergent Phenotypes for Water Holding Capacity across the <italic>Post Mortem</italic> Ageing Period in Porcine Muscle Exudate. *PLoS One* 11, e0150605.
Fisher A.B. (2011) Peroxiredoxin 6: a bifunctional enzyme with glutathione peroxidase and phospholipase A(2) activities. *Antioxid Redox Signal* 15, 831-844.

Fontanesi L., Davoli R., Nanni Costa L., Scotti E. & Russo V. (2003) Study of candidate genes for glycolytic potential of porcine skeletal muscle: identification and analysis of mutations, linkage and physical mapping and association with meat quality traits in pigs. *Cytogenetic and Genome Research* 102, 145-151.

Franco D., Mato A., Salgado F.J., Lopez-Pedrouso M., Carrera M., Bravo S., Parrado M., Gallardo J.M. & Zapata C. (2015) Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress. *J Proteomics* 122, 73-85.

Gagaoua M., Boudida Y., Becila S., Picard B., Boudjellal A., Sentandreu M.A. & Ouali A. (2012) New Caspases' inhibitors belonging to the serpin superfamily: A novel key control point of apoptosis in mammalian tissues. *Advances in Bioscience and Biotechnology* 03, 740-750.

Gagaoua M., Hafid K., Boudida Y., Becila S., Ouali A., Picard B., Boudjellal A. & Sentandreu M.A. (2015a) Caspases and Thrombin Activity Regulation by Specific Serpin Inhibitors in Bovine Skeletal Muscle. *Appl Biochem Biotechnol* 177, 279-303.

Gagaoua M., Terlouw E.M., Boudjellal A. & Picard B. (2015b) Coherent correlation networks among protein biomarkers of beef tenderness: What they reveal. *J Proteomics* 128, 365-374.

Gagaoua M., Terlouw E.M., Micol D., Boudjellal A., Hocquette J.F. & Picard B. (2015c) Understanding Early Post-Mortem Biochemical Processes Underlying Meat Color and pH Decline in the Longissimus thoracis Muscle of Young Blond d'Aquitaine Bulls Using Protein Biomarkers. *J Agric Food Chem* 63, 6799-6809.
Gagaoua M., Laouar K., Hammou I. & Hafid K. (2016) Towards Proteomic Biomarkers of Water
Holding Capacity of Algerian Dromedary One-Humped Camel Meat: proteome basis
concerning the drip loss defect In: *X Annual Congress of the European Proteomics
Association (EuPA)* (ed. by Ozpınar PA), Istanbul, Turkey.

Goll D.E., Thompson V.F., Li H., Wei W. & Cong J. (2003) The calpain system. *Physiol Rev* 83, 731-801.

Gotoh T., Terada K., Oyadomari S. & Mori M. (2004) hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria. *Cell Death Differ* 11, 390-402.

Grabez V., Kathri M., Phung V., Moe K.M., Slinde E., Skaugen M., Saarem K. & Egelandsdal B. (2015) Protein expression and oxygen consumption rate of early postmortem mitochondria relate to meat tenderness. *J Anim Sci* 93, 1967-1979.

Guillemin N., Bonnet M., Jurie C. & Picard B. (2011) Functional analysis of beef tenderness. *J Proteomics* 75, 352-365.

Guillemin N., Jurie C., Renand G., Hocquette J.F., Micol D., Lepetit J. & Picard B. (2012) Different phenotypic and proteomic markers explain variability of beef tenderness across muscles. *Int. J. Biol* 4, 26-38.

Huang H., Larsen M.R., Karlsson A.H., Pomponio L., Costa L.N. & Lametsch R. (2011) Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences. *Proteomics* 11, 4063-4076.

Huff Lonergan E., Zhang W. & Lonergan S.M. (2010) Biochemistry of postmortem muscle - lessons on mechanisms of meat tenderization. *Meat Sci* 86, 184-195.
Hwang I.H., Park B.Y., Kim J.H., Cho S.H. & Lee J.M. (2005) Assessment of postmortem proteolysis by gel-based proteome analysis and its relationship to meat quality traits in pig longissimus. *Meat Sci* 69, 79-91.

Jia X., Ekman M., Grove H., Faergestad E.M., Aass L., Hildrum K.I. & Hollung K. (2007) Proteome changes in bovine longissimus thoracis muscle during the early postmortem storage period. *J Proteome Res* 6, 2720-2731.

Jia X., Hildrum K.I., Westad F., Kummen E., Aass L. & Hollung K. (2006a) Changes in enzymes associated with energy metabolism during the early post mortem period in longissimus thoracis bovine muscle analyzed by proteomics. *J Proteome Res* 5, 1763-1769.

Jia X., Hollung K., Therkildsen M., Hildrum K.I. & Bendixen E. (2006b) Proteome analysis of early post-mortem changes in two bovine muscle types: M. longissimus dorsi and M. semitendinosis. *Proteomics* 6, 936-944.

Jia X., Veiseth-Kent E., Grove H., Kuziora P., Aass L., Hildrum K.I. & Hollung K. (2009) Peroxiredoxin-6--a potential protein marker for meat tenderness in bovine longissimus thoracis muscle. *J Anim Sci* 87, 2391-2399.

Joseph P., Suman S.P., Rentfrow G., Li S. & Beach C.M. (2012) Proteomics of muscle-specific beef color stability. *J Agric Food Chem* 60, 3196-3203.

Kaspric N., Picard B., Reichstadt M., Tournayre J. & Bonnet M. (2015) ProteINSIDE to Easily Investigate Proteomics Data from Ruminants: Application to Mine Proteome of Adipose and Muscle Tissues in Bovine Foetuses. *PLoS One* 10, e0128086.

Kemp C.M. & Parr T. (2012) Advances in apoptotic mediated proteolysis in meat tenderisation. *Meat Sci* 92, 252-259.
Kim N.K., Cho S., Lee S.H., Park H.R., Lee C.S., Cho Y.M., Choy Y.H., Yoon D., Im S.K. & Park E.W. (2008) Proteins in longissimus muscle of Korean native cattle and their relationship to meat quality. *Meat Sci* 80, 1068-1073.

Lametsch R., Karlsson A., Rosenvold K., Andersen H.J., Roepstorff P. & Bendixen E. (2003) Postmortem proteome changes of porcine muscle related to tenderness. *J Agric Food Chem* 51, 6992-6997.

Lametsch R., Roepstorff P. & Bendixen E. (2002) Identification of Protein Degradation during Post-mortem Storage of Pig Meat. *Journal of Agricultural and Food Chemistry* 50, 5508-5512.

Lametsch R., Roepstorff P., Møller H.S. & Bendixen E. (2004) Identification of myofibrillar substrates for μ-calpain. *Meat Science* 68, 515-521.

Laville E., Sayd T., Morzel M., Blinet S., Chambon C., Lepetit J., Renand G. & Hocquette J.F. (2009) Proteome changes during meat aging in tough and tender beef suggest the importance of apoptosis and protein solubility for beef aging and tenderization. *J Agric Food Chem* 57, 10755-10764.

Li C., Zhou G., Xu X., Lundstrom K., Karlsson A. & Lametsch R. (2015) Phosphoproteome analysis of sarcoplasmic and myofibrillar proteins in bovine longissimus muscle in response to postmortem electrical stimulation. *Food Chem* 175, 197-202.

Listrat A., Lebret B., Louveau I., Astruc T., Bonnet M., Lefaucheur L., Picard B. & Bugeon J. (2016) How Muscle Structure and Composition Influence Meat and Flesh Quality. *The Scientific World Journal* 2016, 1-14.

Lomiwes D., Farouk M.M., Wiklund E. & Young O.A. (2014) Small heat shock proteins and their role in meat tenderness: a review. *Meat Sci* 96, 26-40.
Lorenzen C.L., Neely T.R., Miller R.K., Tatum J.D., Wise J.W., Taylor J.F., Buyck M.J., Reagan
J.O. & Savell J.W. (1999) Beef customer satisfaction: cooking method and degree of
doneness effects on the top loin steak. *J Anim Sci* 77, 637-644.

Mao Y., Hopkins D.L., Zhang Y., Li P., Zhu L., Dong P., Liang R., Dai J., Wang X. & Luo X.
(2016) Beef quality with different intramuscular fat content and proteomic analysis using
isobaric tag for relative and absolute quantitation of differentially expressed proteins. *Meat
Sci* 118, 96-102.

Marcos B. & Mullen A.M. (2014) High pressure induced changes in beef muscle proteome: correlation with quality parameters. *Meat Sci* 97, 11-20.

Marino R., Albenzio M., Della Malva A., Caroprese M., Santillo A. & Sevi A. (2014) Changes in meat quality traits and sarcoplasmic proteins during aging in three different cattle breeds. *Meat Sci* 98, 178-186.

Marino R., Della Malva A. & Albenzio M. (2015) Proteolytic changes of myofibrillar proteins in Podolian meat during aging: focusing on tenderness. *J Anim Sci* 93, 1376-1387.

Mekchay S., Teltathum T., Nakasathien S. & Pongpaichan P. (2010) Proteomic Analysis of Tenderness Trait in Thai Native and Commercial Broiler Chicken Muscles. *The Journal of Poultry Science* 47, 8-12.

Miller M.F., Carr M.A., Ramsey C.B., Crockett K.L. & Hoover L.C. (2001) Consumer thresholds for establishing the value of beef tenderness. *J Anim Sci* 79, 3062-3068.

Monastyrskaya K., Babiychuk E.B. & Draeger A. (2009) The annexins: spatial and temporal coordination of signaling events during cellular stress. *Cell Mol Life Sci* 66, 2623-2642.

Montowska M., Rao W., Alexander M.R., Tucker G.A. & Barrett D.A. (2014) Tryptic digestion coupled with ambient desorption electrospray ionization and liquid extraction surface analysis mass spectrometry enabling identification of skeletal muscle proteins in mixtures
and distinguishing between beef, pork, horse, chicken, and turkey meat. *Anal Chem* 86, 4479-4487.

Morzel M., Terlouw C., Chambon C., Micol D. & Picard B. (2008) Muscle proteome and meat eating qualities of Longissimus thoracis of "Blonde d'Aquitaine" young bulls: A central role of HSP27 isoforms. *Meat Science* 78, 297-304.

Muroya S., Ohnishi-Kameyama M., Oe M., Nakajima I., Shibata M. & Chikuni K. (2007) Double phosphorylation of the myosin regulatory light chain during rigor mortis of bovine Longissimus muscle. *J Agric Food Chem* 55, 3998-4004.

Ohta S., Ohsawa I., Kamino K., Ando F. & Shimokata H. (2004) Mitochondrial ALDH2 Deficiency as an Oxidative Stress. *Annals of the New York Academy of Sciences* 1011, 36-44.

Ouali A., Gagaoua M., Boudida Y., Becila S., Boudjellal A., Herrera-Mendez C.H. & Sentandreu M.A. (2013) Biomarkers of meat tenderness: present knowledge and perspectives in regards to our current understanding of the mechanisms involved. *Meat Sci* 95, 854-870.

Ouali A., Herrera-Mendez C.H., Coulis G., Becila S., Boudjellal A., Aubry L. & Sentandreu M.A. (2006) Revisiting the conversion of muscle into meat and the underlying mechanisms. *Meat Sci* 74, 44-58.

Picard B., Berri C., Lefaucheur L., Molette C., Sayd T. & Terlouw C. (2010) Skeletal muscle proteomics in livestock production. *Brief Funct Genomics* 9, 259-278.

Picard B., Gagaoua M., Kammoun M., Terlouw C., Hocquette J.F. & Micol D. (2013) Biomarkers of beef tenderness in young bulls of three breeds. In: *Proceedings of the 59th International Congress of Meat Science and Technology*, p. 4, Izmir, Turkey.
Picard B., Gagaoua M., Micol D., Cassar-Malek I., Hocquette J.F. & Terlouw C.E. (2014) Inverse relationships between biomarkers and beef tenderness according to contractile and metabolic properties of the muscle. *J Agric Food Chem* 62, 9808-9818.

Platter W.J., Tatum J.D., Belk K.E., Koontz S.R., Chapman P.L. & Smith G.C. (2005) Effects of marbling and shear force on consumers' willingness to pay for beef strip loin steaks. *J Anim Sci* 83, 890-899.

Polati R., Menini M., Robotti E., Millioni R., Marengo E., Novelli E., Balzan S. & Cecconi D. (2012) Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing. *Food Chemistry* 135, 2052-2069.

Przybylski W. & Hopkins D. (2015) *Meat Quality: Genetic and Environmental Factors*. CRC Press. Ed Wieslaw Przybylski, PhD, David Hopkins, PhD. 333-365.

Pulford D.J., Fraga Vazquez S., Frost D.F., Fraser-Smith E., Dobbie P. & Rosenvold K. (2008) The intracellular distribution of small heat shock proteins in post-mortem beef is determined by ultimate pH. *Meat Sci* 79, 623-630.

Sawdy J.C., Kaiser S.A., St-Pierre N.R. & Wick M.P. (2004) Myofibrillar 1-D fingerprints and myosin heavy chain MS analyses of beef loin at 36 h postmortem correlate with tenderness at 7 days. *Meat Sci* 67, 421-426.

Sentandreu M.A., Coulis G. & Ouali A. (2002) Role of muscle endopeptidases and their inhibitors in meat tenderness. *Trends in Food Science & Technology* 13, 400-421.

Shackelford S.D., Wheeler T.L., Meade M.K., Reagan J.O., Byrnes B.L. & Koohmaraie M. (2001) Consumer impressions of Tender Select beef. *Journal of Animal Science* 79, 2605-2614.

Sierra V. & Olivan M. (2013) Role of mitochondria on muscle cell death and meat tenderization. *Recent Pat Endocr Metab Immune Drug Discov* 7, 120-129.
Thomas K.J., McCoy M.K., Blackinton J., Beilina A., van der Brug M., Sandebring A., Miller D., Maric D., Cedazo-Minguez A. & Cookson M.R. (2011) DJ-1 acts in parallel to the PINK1/parkin pathway to control mitochondrial function and autophagy. *Hum Mol Genet* 20, 40-50.

Tian X., Wu W., Yu Q., Hou M., Jia F., Li X. & Dai R. (2016) Quality and proteome changes of beef M.longissimus dorsi cooked using a water bath and ohmic heating process. *Innovative Food Science & Emerging Technologies* 34, 259-266.

Wu W., Yu Q.-Q., Fu Y., Tian X.-J., Jia F., Li X.-M. & Dai R.-T. (2015) Towards muscle-specific meat color stability of Chinese Luxi yellow cattle: A proteomic insight into post-mortem storage. *J Proteomics*. Nov 3, 1874-3919.

Zapata I., Zerby H.N. & Wick M. (2009) Functional proteomic analysis predicts beef tenderness and the tenderness differential. *J Agric Food Chem* 57, 4956-4963.

Zhao Y.M., Basu U., Dodson M.V., Basarb J.A. & Guan le L. (2010) Proteome differences associated with fat accumulation in bovine subcutaneous adipose tissues. *Proteome Sci* 18, 8-14.