Proteomics approaches – their potential for answering complex questions in meat science research

Abstract There is a consistent and growing need for livestock and meat producers to understand the factors that affect fresh meat quality and value. New information can be used to make precise changes in management or genetic selection to improve the ability to efficiently produce the high-quality meat that customers and consumers expect. The molecular composition of meat can explain the variation in the meat quality phenotype. The molecular phenotype includes the profile of all molecules in the proteome, metabolome, and lipidome. Because a high proportion of meat quality traits are affected by structural and metabolic proteins, identifying the proteomic phenotype associated with high-quality meat production will inform the development of strategies to consistently produce these products. There is a complexity in interpreting early post-mortem, post-rigour, and post-ageing muscle proteomes, and these phenotypes are distinct from those observed in living muscle. Investigations using proteomic tools must carefully ask direct questions in the context of post-mortem muscle. New technologies provide the opportunity to measure entire protein profiles, and even more information can be learned by defining the proteome of fractions of proteins separated based on their solubility and observed changes during the post-mortem conversion of muscle to meat. Targeted studies of how specific proteins that are known to directly affect meat quality, such as myoglobin and calpain, demonstrate that modifications of these proteins can affect their activity and how they contribute to meat quality. A holistic view of the meat proteome and recognising that it is specifically different than the muscle proteome will provide the meat science community with a starting point to use proteomic and other omic technologies to address these complex issues. This review aims to provide an overview of the current state-of-the-art in proteomics s in meat science research, highlighting recent advancements and perspectives for future research. HIGHLIGHTS Interpreting data on protein interactions in meat is challenging due to the vastly different intracellular environment compared to muscle. Omics approaches provide insights into the molecular processes underlying meat quality. Targeted proteomic approaches need to be considered to provide a fuller understanding of the changes important to meat quality.


Introduction
The meat industry has faced growing challenges in meeting the increasing necessity for the efficient production of high-quality meat products.Production of high-quality protein is critical to ensure adequate food to meet the nutritional demands of an ever-growing population.Meat is an important contributor to that need.Furthermore, the future sustainability of this industry and continued supply of high-quality animalsource protein depends on the continued production of meat that is safe, nutritious, and desirable by consumers.A decline in any of these attributes will cause a loss of demand.Consumer acceptance of meat depends on a positive eating experience, including tenderness and flavour profile of meat (Guelker et al. 2013) and colour (Ramanathan et al. 2022).Production of product that is not purchased or consumed certainly has a negative impact on the sustainability of the meat production system (Ramanathan et al. 2022).Therefore, ensuring quality is an important part of the larger sustainability landscape in the livestock and food industry.

The complex landscape of fresh meat quality
The quality aspects of fresh meat are particularly challenging to predict and manage.Scientists have been trying for decades to definitively solve challenges to fresh meat quality (Huff- Lonergan and Lonergan 2005;Huff Lonergan et al. 2010;Huang et al. 2020;Purslow et al. 2021;Warner et al. 2021Warner et al. , 2022)).Quality issues like lack of tenderness, poor water-holding capacity, and inferior and/or unstable colour are all issues that plague the industry and defy easy solutions and development of prediction technologies.Much of the reason that solutions to questions and challenges have not been easy to find is because meat is derived from skeletal muscle tissue.Skeletal muscle is a dynamic tissue subject to many changes and influences.Animal differences and the responses of the tissue to external influences like animal management from early life through harvest and handling of the product are sources of variation in fresh meat quality.For example, many animal production factors regulate skeletal muscle growth and development, including genetics and nutrition (Hochmuth et al. 2022;Schulte et al. 2023).Another complicating factor is that during the conversion of muscle to meat, the tissue responds to changes in the cellular environment that occur early post-mortem.Both ante and post-mortem conditions influence cellular and tissue responses to the conversion of muscle to meat (Purslow et al. 2021;Hochmuth et al. 2022).This cascade of events has long-term ramifications for the quality of the product.
Truly mapping what is happening in early postmortem muscle/meat is key to developing solutions to quality issues (Purslow et al. 2021).It is well documented that protein variations and their function contribute greatly to the variation in meat quality.Therefore, the definition of these changes and the factors that influence the rate and extent of protein changes are necessary to make improvements.Scientists have been trying to do just that for decades but have been stymied by the complexities of not just the structure of the tissue, but by the complexity of the changes in post-mortem metabolism that occur (Huff Lonergan et al. 2010;Chauhan and England 2018;Chen et al. 2019;Matarneh et al. 2021;Zou et al. 2023).We can learn much about muscle structure and metabolism from biomedical studies focusing on living muscle (Melby et al. 2020;Liu et al. 2022;Melby et al. 2023;Smith et al. 2023).However, the dynamics of the early post-mortem environment can create situations unlike any observed in living muscle (Huff Lonergan et al. 2010).This early post-mortem environment sets up a cascade of events that can differentially impact changes in proteins and metabolites at later ageing times (Huff Lonergan et al. 2010;Schulte et al. 2021Schulte et al. , 2023)).These changes not only impact the product, they also introduce complexity in interpreting early post-mortem, post-rigour, and post-ageing muscle proteomes.One example of complicating factor when interpreting the proteome of aged meat is the presence of degraded proteins.When using whole (unfractionated) muscle protein preparations, evaluating the absolute abundance of proteins does not give a complete picture of the changes that may have occurred in aged products.For example, proteolysis produces peptides that may represent the abundance of the parent protein in the total skeletal muscle preparation, but not reflect the form or the solubility of the protein or degradation product at that post-mortem time (Carlson et al. 2017).Some studies have attempted to address the issue of the dynamics of the changes that occur in the proteome either by documenting the changes in the whole muscle proteome (Lamri et al. 2023a;2023b), or by evaluating the subproteomes of soluble proteins in addition to the whole muscle preparations (Schulte et al. 2021;Johnson et al. 2023;Schulte et al. 2023).Existing databases established using living muscle are powerful and useful (Kiyimba et al. 2022) and undoubtedly provide valuable information but do not always provide the specific context completely necessary to apply omics technologies to answer questions about fundamental explanations for variation in fresh meat quality.These databases may not always account for proteolysis, localisation (including solubility) changes, and other post-mortem alterations to muscle proteins.A fresh meat protein context is necessary to develop bioinformatics applications to advance efforts to efficiently produce high-quality meat.
In recent years, advancements in analysis tools have aided the attempts to understand the complexity of the events involved in the conversion of muscle to meat.Omics approaches, particularly proteomics and metabolomics, are being recruited to provide comprehensive insights into the molecular and genetic processes underlying meat quality (Kiyimba et al. 2022;Ramanathan et al. 2023).Proteomics techniques are focused on trying to understand the proteome of an organism or tissue.The proteome was first defined as the 'total protein complement of the genome' (Wasinger et al. 1995).Proteomics techniques hold great promise for identifying key proteins that are critical to understanding as the industry searches for solutions to quality issues.Broadly speaking, proteomics involves the identification and relative quantification of proteins and particularly in meat, protein fragments.Another consideration is that the scientific inquiry of traits that impact meat quality must not only rely on the aspects of proteomics that enable the identification of proteins and protein fragments, but it must also more deeply consider the multitude of post-translational modifications that influence meat's sensory and nutritional properties.
Because of the challenges posed by the complex nature of meat samples, proteomics, and metabolomics have been increasingly applied to meat science research to address a wide range of questions related to meat quality, safety, and sustainability.The intense interest from many scientists in harnessing the power of 'omics' techniques to answer the complex problems of meat science has resulted in a plethora of studies, particularly in proteomics.These studies have been the source of many extensive reviews that attempt to harness the vast amount of knowledge that exists in the proteomics of meat quality (Huang et al. 2020;Picard and Gagaoua 2020;Gagaoua et al. 2021a;2021b).The aforementioned studies, reviews, and others like them (Purslow et al. 2021;Schulte et al. 2021;Zhu et al. 2021;Hochmuth et al. 2022;Johnson et al. 2023;Schulte et al. 2023;Zhu et al. 2023;Lamri et al. 2023a;2023b) have been valuable in providing some insight into post-mortem muscle changes linked to meat quality.Important studies and reviews like these reinforce the fact that the complexity of the problems facing prediction and ultimately controlling meat quality is rooted in our need to understand the dynamics of living and post-mortem muscle changes.Identifying changes to the proteome that are unique to a meat quality/trait 'phenotype' provides a needed avenue to develop tools to identify quality traits in products.Predicting quality development is a more complex question.However, proteomics of ante-mortem or at-death/early post-mortem muscle can aid in providing clues to predict how quality traits may subsequently develop and be detected.

The challenge and evolution of proteomicsan overview
To address the aforementioned complexity of meat quality traits, there is a need to understand the variation in the diversity and abundance of proteins and their forms in early post-mortem muscle and meat -especially regarding meat quality phenotypes impacting value.The study of the early post-mortem muscle and the meat proteome has emerged as an extraordinarily valuable area of inquiry in meat science to address the critical need to understand changes/differences in proteins that impact meat quality (Huang et al. 2020;Purslow et al. 2021;Zhu et al. 2023).Proteomics is a term that encompasses analytical techniques that can be used to study the complex complement of proteins that exist at a particular point in time in a sample.By nature, this definition includes numerous protein analysis methods.Over the course of the years, researchers have used many techniques to understand the complex changes in meat that correspond to observed quality phenotypes.Examples include one-dimensional SDS-PAGE, western blotting, ELISA, dot-blot, and reverse phase protein arrays (RPPA) based techniques, (Huff- Lonergan et al. 1996;Huff Lonergan et al. 2010;Lametsch et al. 2011;Anderson et al. 2012;Gagaoua et al. 2017Gagaoua et al. , 2018aGagaoua et al. , 2018b;;Purslow et al. 2021).These types of assays have allowed many important discoveries to be made, however, they are sometimes not easy to make quantifiable comparisons as many are semi-quantifiable (for example western blotting), nor are they amenable to examining changes in upwards of hundreds of proteins between samples.Over the past 20 years, there have been dramatic increases in the analytical capability to study the proteome by attempting to study the entire set of proteins expressed by a cell, tissue, or organism (Ohlendieck 2011;Faria et al. 2017;Cervone et al. 2023).These advancements in proteomic techniques have also been used to identify differentially abundant proteins/peptides in post-mortem muscle tissue/meat (Picard and Gagaoua 2017;Picard et al. 2018;Huang et al. 2020;Johnson et al. 2023;Schulte et al. 2023;Lamri et al. 2023a;2023b).
An example of an improvement that improved the ability to make comparisons across samples is the introduction of the use of fluorescent dyes (CyDyes) in two-dimensional gel electrophoresis (Marouga et al. 2005).This innovation was extraordinarily useful in efficiently identifying changes in multiple samples (Lametsch et al. 2003;Anderson et al. 2012;Di Luca et al. 2013;Anderson et al. 2014;Carlson et al. 2017).Known as 2D DIGE (Two-Dimensional Difference In Gel Electrophoresis), protein samples (typically up to three) from different treatments or tissues are labelled with different fluorescent dyes before they are mixed and run on a gel.These dyes covalently bind to the lysine residues of proteins.The dyes used emit distinct fluorescent signals.This labelling strategy enables multiple samples to be analysed simultaneously within the same gel by selectively exciting the dyes and imaging the resulting fluorescence.The approach facilitates more accurate comparisons of the differences in the abundance of labelled proteins between the samples.Typically, two samples with different dyes are run at a time.To account for gel-to-gel variations, each gel also contains an internal standard that is labelled with a third dye.The internal standard consists of a mixture of equal amounts of all the samples being analysed in the experiment and aids in normalising the protein quantities across different gels.The power of this technique over traditional two-dimensional gels is that it allows more efficient elucidation of proteins that are differentially abundant between samples because multiple (up to three) samples are identity tagged and loaded on a single gel, thus reducing some gel to gel variation.A critical next step is picking the differentially abundant protein spots from the gel and identifying them using mass spectrometry methods.This method is laborious, time-consuming, and has lower thruput compared to approaches based solely on LC-MS.One benefit of gel-based methods is that they can be more selective and, as will be described later, can have distinct advantages when addressing more targeted research questions (Anderson et al. 2012;AndersonLonergan and Huff-Lonergan, 2014;Carlson et al. 2017).
Currently, LC-MS approaches are used much more extensively to study the proteome of biological systems (Melby et al. 2023;Shuken 2023).This is true as well in meat science (Johnson et al. 2023;Zhai et al. 2023;Zhu et al. 2023;Lamri et al. 2023b).LC-MS based proteomic methods are increasingly being used and have yielded important discoveries regarding key proteins that need to be considered in investigations designed to resolve elusive questions revolving around meat quality.Much of the current work utilises discovery-based approaches to proteomics.Collectively, these approaches are often referred to as 'shotgun proteomics' or bottom-up proteomics.The approaches depend on digesting proteins into peptides using (most commonly) trypsin, although other proteases like Lys-C and chymotrypsin can be used (Giansanti et al. 2016).The resulting peptides are separated using high-performance liquid chromatography and then injected into a mass spectrometer.Downstream, the ionised peptides are analysed for their mass to charge ratio (m/z), and the precursor ions are fragmented by methods using MS/MS for analysis.Fragmented peptides are further analysed for their m/z (for a detailed discussion of the methodology, see (Shuken 2023).This method provides information about the amino acid sequences of the peptides, and using databases can provide the identity and relative abundance of the parent proteins (Faria et al. 2017;Shuken 2023).This type of proteomic analysis can be classified into label-free or labelbased (using isobaric or isotopic tags).Recent improvements in instrumentation and chemistry have allowed more widespread usage of isobaric tags.These tandem mass tag (TMT) approaches allow high throughput comparisons of multiple samples in a mass spectrometry analysis run (Rauniyar and Yates 2014).Indeed, this approach has been used in many studies from clinical trials in animal science (Chen et al. 2023) through meat science studies (Johnson et al. 2023;Schulte et al. 2023).
These bottom-up approaches have been extraordinarily useful and are very efficient in identifying hundreds of proteins in samples, yet they are not as amenable to hypothesis-driven inquiry as other methods (Faria et al. 2017;Cervone et al. 2023).Bottom-up proteomics approaches provide very broad results and typically do not lend themselves as easily to studies with hypotheses related to changes in specific proteins (Faria et al. 2017).Bottom-up proteomic experiments tend to address more non-specific questions, such as identifying the increased or decreased abundance of classes of proteins.While very useful, this information can be more complex and harder to home in on specific proteoforms (Jungblut et al. 2008;Schluter et al. 2009) or examine potential biomarkers.Another issue with bottom-up or shotgun proteomics is that the peptides produced and analysed are present in abundance relative to the amount of the parent protein in the sample (Shuken 2023).In any proteomic experiment, regardless of the method, overabundant proteins can alter the ability to detect other proteins (Grubbs et al. 2015).In whole muscle preparations, this makes proteins like actin, myosin, and titin in skeletal muscle the most likely to be identified and can allow less abundant proteins to be overshadowed and not as easily detected or represented in the results, thus potentially overlooking their importance in the study.Many studies have identified large numbers of proteins that are present, yet specific answers to questions in meat science are not as readily available as researchers would like.It is now becoming apparent that while there is immense value in the bottom-up approaches that have and are being used, more proteomic studies that focus on hypotheses around changes in key proteins are needed to build on the bottom-up approaches are needed.
They provide added progress in understanding complex problems in meat science and in potentially developing viable biomarkers for some characteristics.One key to understanding the variation in meat quality development is the variation in the proteins in muscle themselves (post-translational modifications, protein folding, proteolysis etc.).Therefore, the concept of 'proteoform' is important to be mindful of in meat quality studies.

Proteoforms
Researchers working with living systems have discovered that understanding the genome is just the beginningwhile biological systems are regulated by the proteins coded for by the genome, the variation at the protein level is quite varied and complexmuch more than can be accounted for by specific genes (Schluter et al. 2009).Therefore, it is key to understand the sources of variation in a tissue's protein population (Jungblut et al. 2008).This variation arises from many different sources, including post-translational modifications such as protein folding, phosphorylation, glycosylation, and lipidation as well proteolytic processing (Manzoni et al. 2018).In 2013 it was recognised that no term was truly appropriate to describe these protein variations (Smith et al. 2013).The argument was made that isoform is not appropriate as many organisations use the term 'isoform' differently.For example, the International Union of Pure and Applied Chemistry (IUPAC) uses 'isoform' to refer to genetic differences and does not specifically include protein diversity introduced by post-translational modifications.The term 'proteoform' has been proposed to refer to all the different forms a protein from one gene can be found to exist in (Smith et al. 2013(Smith et al. , 2019(Smith et al. , 2021)).This term includes the many different molecular forms of a protein that can be produced from splice variants, isoforms, post-translational modifications etc. (CarbonaraAndonovski and Coorssen, 2021).Even redox changes with the proposed name of 'oxiform' has been proposed to be part of the lexicon describing the proteoform (Cobley 2023).The recognition that just identifying proteins in a sample, while useful, is not enough without considering the multiple forms they may be in.This is true for living muscle as well as post-mortem muscle/meat.This recognition has led to the need to develop/use more top-down approaches to identifying the diversity of proteoforms and their contributions to the phenotypes being studied (Faria et al. 2017;Marcus et al. 2020;Carbonara et al. 2021).
As described in the previous passage, there are physiologically significant modifications of proteins in muscle and meat that can fall under the term proteoform.These include phosphorylation (Lametsch et al. 2003;Gagaoua et al. 2021a), nitrosylation (Warner et al. 2005(Warner et al. , 2010)), acetylation (Wang et al. 2021) and ADP ribosylation (Leutert et al. 2018).Some modifications appear to contribute to early post-mortem metabolism and the rate and extent of molecular events contributing to meat quality development.Some events and environments are created in early post-mortem muscle that are out of the context of living muscle but can dictate molecular changes and meat quality.Certainly, accumulation of lactate, a pH decline, loss of antioxidant potential, accumulation of lipid oxidation products, and an increase in ionic strength affect protein structure and function.Post-mortem protein degradation, regardless of the causative agent, changes the context and function of proteins in meat.Thus, understanding the proteoform of living muscle and post-mortem muscle/meat is important Targeted protein phosphorylation in perimortem and potentially early post-mortem muscle can alter energy metabolism and the rate and extent of changes in meat quality traits (Gagaoua et al. 2021a).New methods and higher resolution instrumentation will provide the opportunity specifically define phosphorylated proteins in bottom-up and top-down approaches.The stage is set to ask specific questions regarding how these changes can be used as biomarkers.Developing strategies to quantify proteoforms (for example, entire molecule phosphorylation) will be important to document specific modifications.
Another example of modifications that need to be considered more carefully is protein nitrosylation.Published data show nitrosylated proteins are detected in post-mortem muscle (Warner et al. 2005;Huff Lonergan et al. 2010;Warner et al. 2010;Liu et al. 2019).Nitrosylation does change structure and function.However, detailed information on specific proteins that are modified, when they are nitrosylated, and what sites are modified is not readily available.There are some indications that nitrosylation does influence protein function in model systems (Liu et al. 2019;Lu et al. 2023).Further, though it appears that while proteins may be nitrosylated in the early postmortem period, the specific modifications and their impact on protein function are not clearly defined.

Targeted proteomics
Analysing the complexity of proteoforms requires complementary tools.In contrast to identifying proteins from the peptide level, as with bottom-up approaches, top-down proteomics can add to the richness of knowledge regarding the phenotypes of interest.Top-down proteomics focuses on identifying proteoforms that are unique by analysing intact proteins with mass spectrometry (Melby et al. 2020(Melby et al. , 2021(Melby et al. , 2023)).The significant advancement in the development of mass-spectrometry methods has enabled the use of this technology to study proteins (Toby et al. 2016;Melby et al. 2020Melby et al. , 2023) ) including proteins and protein modifications important to meat science (Zhai et al. 2023).However, analysing complex samples using these techniques is still difficult because of the wide dynamic range (Carbonara et al. 2021).Methods such as prefractionation to decrease sample complexity can aid in allowing these techniques to be used (Melby et al. 2021).In addition to top-down proteomics using mass spectrometry, approaches that not only use pre-fractionation, and gel-based techniques, such as two-dimensional gel electrophoresis coupled with DIGE and/or specific protein staining and western blotting, can be used (Faria et al. 2017;Marcus et al. 2020;Carbonara et al. 2021;Cervone et al. 2023).These techniques lend themselves to hypothesis-based research and can draw from the rich data from numerous bottom-up studies to guide approaches being used.Utilisation of pre-fractionation, enrichment, and top-down methods will generate a clear picture of the post-mortem and post-rigour proteomes that are distinctly different than those of living muscle.Generating databases with those data will provide tools to improve management and genetic practices to improve meat quality.Regardless, there are many examples of targeted proteomic studies that when considered in tandem with bottom-up proteomics studies can yield important insights.

Case studies connecting Targeted proteomics and Post-Translational modifications to meat quality
Targeted proteomic studies have been employed to determine the extent to which modifications of key proteins influence their function and, ultimately, meat quality.This approach requires much knowledge about the protein and its functions in muscle and meat.The rate and extent of post-mortem glycolysis influences meat quality and phosphoglucomutase participates in that pathway.There is evidence that phosphoglucomutase À1 (PGM-1) is phosphorylated.Two other proteins that have been studied extensively are myoglobin and calpain.Calpains and myoglobin are sensitive to their environment, but modifications of these proteins in muscle, meat, and model systems show that modifications can be a source of variation in their function and their contribution to meat quality.These observations provide evidence that post-translational modifications of key proteins should be documented.

Phosphoglucomutase -1
Pre-fractionation of samples followed by analysis using 2-D DIGE and selective phosphoprotein staining has been used to identify changes in the proteoforms of beef longissimus muscle of different tenderness classifications (Anderson et al. 2012(Anderson et al. , 2014)).Anderson et al. (2014) showed that specific spots in a 'train' of spots in a 2D-DIGE experiment were all identified as isoforms of phosphoglucomutase-1 (PGM-1) when they were picked and identified using MS/MS techniques.Individual spots in the 'train' of PGM-1 were differentially abundant between the tenderness classifications.Phosphoprotein staining and western blotting confirmed the spots contained a differentially phosphorylated protein.Subsequent analysis showed that the least phosphorylated form of the protein was associated most strongly with the least tender beef, suggesting not simply that amount of PGM-1, but also its state, in this case, degree of phosphorylation, must be considered.In this case, examination of the occurrence of PGM-1 on a onedimensional gel would not have revealed the differences that were noted in the two-dimensional gel as all isoforms would have migrated to the same location.This is an example of the need to consider not only the abundance of protein, but also the form in which it exists.

Myoglobin
Myoglobin is a monomeric sarcoplasmic haemcontaining protein in muscle and meat.The amount and state of myoglobin are primarily responsible for the colour of meat products (Mancini and Hunt 2005).In most cases, the state of myoglobin implies an oxidation state or gas bound to the iron in the haem porphyrin ring structure.Recently it has become evident that modifications of the protein that occur perimortem, early post-mortem, or post-rigour can influence the stability of the molecule and, therefore, colour stability reviewed by (Ramanathan, Suman et al. 2020;Ramanathan, Hunt et al. 2020).

Myoglobin phosphorylation
Myoglobin is known to be phosphorylated, and first reported phosphorylation of beluga whale myoglobin, specifically at serine 117, was reported in 2004 (Stewart et al. 2004).Phosphoprotein specific ProQ staining was used to identify 23 phosphorylated proteins, including myoglobin in porcine sarcoplasmic protein (Huang et al. 2011;Lametsch et al. 2011).Phosphorylation of ovine myoglobin was documented, and phosphorylation of myoglobin, in addition to some other targets, was associated with less tender lamb meat (Li et al. 2017).
Recently several lines of investigation have demonstrated that modification of the myoglobin protein can alter the susceptibility of myoglobin to oxidation and, therefore, colour stability.On a 2-D gel, four different spots of myoglobin were detected (Canto et al. 2015).Those authors proposed that phosphorylation was the reason for the diversity in observed spots and that phosphorylation of myoglobin explained variation in colour stability.Using phosphatase and protein kinase inhibitors, Li et al. (2017) generated different phosphorylation levels of myoglobin in lamb and showed a positive correlation between myoglobin phosphorylation and oxidation of myoglobin to form metmyoglobin.Phosphorylation of serine 133 in myoglobin has been proposed to negatively affect myoglobin stability and meat colour stability (Li et al. 2018).In an indepth study, Wang et al. (2021) documented the phosphorylation of serine (positions 58, 108, 121, 132), tyrosine (position 103), and threonine (position 34, 51, 67,70) of bovine myoglobin.Those authors proposed that phosphorylation of threonine 67 would influence the function of the distal histidine at position 64 and perhaps limit the oxygen binding ability of myoglobin.

Modification of myoglobin by adduction of lipid oxidation products
Adduction of lipid oxidation products, specifically 4hydroxy-2-nonenal (HNE) accelerated oxidation of equine myoglobin (Faustman et al. 1999).This effect is somewhat pH dependent, as it may not be as pronounced when exposure occurs at a lower pH.Equine cardiac oxymyoglobin oxidation rate increased in the presence of HNE; this observation was at least partially due to the observation that the exposed myoglobin was a poorer substrate for enzymatic metmyoglobin reduction (Lynch and Faustman 2000).Six histidine residues of bovine myoglobin that were rapidly adducted by HNE have been described (Alderton et al. 2003).These included the proximal (position 93) and distal (position 64) histidines attending the haem group.Importantly, these modifications were associated with a destabilisation of the molecule and an increased susceptibility to haem iron oxidation.
Because histidine is a primary target for HNE adduction, porcine myoglobin -with nine histidine residues -is possibly less likely to be modified than bovine myoglobin -with 13 histidine residues (Suman and Joseph 2013).HNE incubation in conditions similar to post-rigour muscle renders bovine myoglobin more susceptible to oxidation to metmyoglobin than porcine myoglobin (Suman et al. 2006)).HNE modified four histidine residues in bovine myoglobin, and only two residues were modified by HNE in porcine myoglobin.The more discoloration-susceptible bovine myoglobin had diadducts of HNE, whereas porcine myoglobin had monoadducts of HNE.The subtle difference in sequence and amino acid position in myoglobin from different species changes modification, susceptibility to oxidation, and meat colour stability.Lysine HNE alkylation of bovine myoglobin at positions 78 and 79 was detected in aged beef, suggesting that HNE alkylation occurs post-rigour when meat colour stability is declining (Wang et al. 2021).An increase in the production of lipid oxidation products does coincide with alkylation and a decrease in colour stability.
The extent to which modifications of myoglobin affect colour and colour stability is still not clarified.Wang et al. (2021) demonstrated numerous postmortem modifications in myoglobin extending over 21 days post-mortem.These included HNE alkylation, phosphorylation, acetylation, methylation, and carboxymethylation.Some subtle changes may affect myoglobin colour stability like those already identified.These small changes will help determine heretofore undefined sources of variation in meat colour and colour stability.

Calpain system
Ageing meat at refrigerated temperatures after completion of rigour results in a decrease in shear force and an improvement in tenderness in beef, pork, and lamb (Huff Lonergan et al. 2010;Schulte et al. 2019).A great deal of evidence supports the hypothesis that protein degradation in post-mortem muscle is responsible for the so-called resolution of rigour and improved tenderness of meat products (Geesink et al. 2006;Warner et al. 2010;2021).Many of these changes are attributed to proteolysis by different proteases, but the prevailing evidence points to calpain activity.When calpain-1 is incubated with myofibrils, many of the same changes are recognised.Notably degradation of titin, nebulin, vinculin, filamin, desmin, and troponin-T (Koohmaraie 1992;Huff-Lonergan et al. 1996;Koohmaraie 1996).The rate and extent of the change in structural integrity are specifically linked to the degradation of structural proteins associated with the myofibril (troponin-T, filamin, vinculin, desmin, nebulin, titin) (Koohmaraie et al. 1995;Taylor et al. 1995;Rowe et al. 2004b, a) Several proteinase systems likely contribute to these changes, but there is considerable evidence in the published literature that implicates the calpain system (specifically calpain-1) as a primary causative agent in much of the proteolysis and improvement in tenderness (reviewed by (Warner et al. 2021).Incubating myofibrils with calpains results in similar proteolytic patterns of desmin, titin, nebulin, and troponin-T that are observed in post-mortem meat (Huff- Lonergan et al. 1996;Geesink and Koohmaraie 1999;Veiseth et al. 2001;Geesink et al. 2006).Calpastatin (an endogenous calpain inhibitor) limits calpain activity, and post-mortem proteolysis is limited under conditions with greater calpastatin activity (Koohmaraie 1992(Koohmaraie , 1994(Koohmaraie , 1996;;Lonergan et al. 2001).Furthermore, adding calpastatin to calpain and myofibril incubations slows down or arrests the degradation of those proteins (Maddock et al. 2005;Carlin et al. 2006).Activating the calpain in pre-or post-rigour muscle with an infusion or injection of calcium chloride stimulates more rapid proteolysis of proteins but still a similar profile (Koohmaraie and Shackelford 1991;Wheeler et al. 1992;Harris et al. 2001).Finally, oxidising conditions in post-rigour meat slow down the activation of calpain and decrease the production of known degradation products (Rowe et al. 2004a(Rowe et al. , 2004b)), and oxidation of calpain in vitro decreases activity and degradation of myofibrillar proteins (Carlin et al. 2006).It is therefore important to recognise the molecular modifications of calpains that can contribute to variation in activation and activity against structural protein substrates in post-mortem muscle.

Oxidation of calpains and adduction with lipid oxidation products
Oxidation of lipids and proteins occurs early in post-mortem and post-rigour meat.These events and their products can directly affect proteins, most notably proteolysis (Rowe et al. 2004a(Rowe et al. ,2004b) ) and associated quality traits.It is well understood that ante-and perimortem events can set the stage for oxidation in post-mortem muscle and meat.In fact, cattle diets high in sulphur (which can create an oxidising environment in post-rigour meat) resulted in decreased calpain-1 activation and post-mortem proteolysis of troponin-T in beef (Pogge et al. 2014).Oxidising conditions created in beef with high oxygen modified atmosphere packages decreased tenderness by promoting cross-linking between myosin heavy chain and titin (Kim et al. 2010).Injection of CaCl 2 in early postmortem muscle increased the concentration of lipid oxidation products (Harris et al. 2001), which slowed the rate of protein degradation and improvement in tenderness.Most of these oxidising events can be mitigated by including antioxidants like Vitamin E (Harris et al. 2001, Rowe et al. 2004a,2004b).These observations support the hypothesis that even low oxidation levels can influence proteolysis and that increasing the antioxidant capacity in meat has merit in improving tenderness.Low levels of oxidation may be the cause of some of the unexplained variations in proteolysis and tenderness that have been observed in meat.Rowe et al. (2004aRowe et al. ( ,2004b) ) showed that increased sarcoplasmic and myofibrillar protein carbonylation resulted in less activation of calpain, less protein degradation, and less tenderisation of beef.
Carbonylation of calpain-1 specifically changes calpain activation and activity (Zhai et al. 2023).In addition, increased carbonylation and decreased sulfhydryl content change the calpain-1 structure, specifically decreasing the alpha helix structure (Liu et al. 2021).These recent reports provide convincing evidence that the carbonylation of specific proteins is a source of variation in meat tenderness.Muscle tissue contains mono-and poly-unsaturated fatty acids, and oxidation during the post-mortem period can form various aldehydes and ketones.This can alter protein functionality, affecting meat quality attributes such as colour, tenderness, and flavour.A recent study showed that rate of post-mortem muscle proteolysis was slower and meat tenderness was less in meat under high-oxygen packaging (greater oxidation) than in anaerobic packaging during cold storage (Vierck et al. 2020).Malondialdehyde (MDA), hexenal, and (HNE) are lipidoxidation-derived aldehydes that have been identified in fresh meat under aerobic packaging (Lynch and Faustman 2000) and the reactivity of these aldehydes increase with the increase in carbon number (Zhai et al. 2019).The effect of these lipid oxidation products (MDA, hexenal, and HNE) on purified porcine calpain-1 activity was recently investigated (Zhai et al. 2023).In general, the calpain-1 activity was decreased by the incubation with 100, 500, or 1000 mM of MDA, hexenal, or HNE compared with controls.However, the calpain-1 activity increased (compared to the control) after incubation with 100 mM MDA but was not different than the control after incubation with 500 mM and 1000 mM MDA.The hexenal and HNE incubations decreased calpain-1 activity at all three concentrations (100 mM, 500 mM, and 1000 mM) compared to the control (0 mM).Specifically, calpain-1 activity decreased with the increase in the concentration of hexenal and HNE from 100 mM to 1000 mM.The change in calpain-1 activity by MDA, HNE, and hexenal was concomitant with the protein modification.Schiff base type adducts were detected in the MDA-treatment group on the side-chain groups of lysine, histidine, and asparagine residues in calpain.Perhaps the most notable modification was detected at histidine 272 in the active site.Both the treatment and control groups had HNE adducts on various amino acids in calpain-1.Hexenaladducted peptides were not identified in that study.Zhai et al. (2023) demonstrated that calpain-1 responds differently to oxidants in early post-mortem muscle and aged meat.However, these demonstrations were made under physiological pH of 7.4 in an isolated system without calpastatin.In meat systems, the ultimate pH varies depending on post-mortem metabolic pathways and affects meat quality.Furthermore, calpastatin inhibits calpain-1 activity and is an integral component of the overall calpain system in meat.Therefore, it is important to understand the implications of pH variations and the presence of calpastatin on the interaction between calpain-1 and carbonylation.Indeed, Carlin et al. (2006) demonstrated that pH and calpastatin changed the calpain-1 response to exposure to hydrogen peroxide, showing that oxidation of the calpain/calpastatin complex could promote calpain activation.The current gap in the knowledge regarding how pH and calpastatin can influence the susceptibility of calpain to modification by lipid oxidation products represents a lack of understanding of how calpain modifications in post-mortem and post-rigour muscle.
Schiff base type adducts of MDA on the side-chain groups of glutamine, lysine, asparagine, and arginine were detected in calpain-1 that was isolated from very early post-mortem muscle and was never exposed to the oxidation products in vitro (Zhai et al. 2023).This novel observation supports the hypothesis that oxidation-induced modifications of calpain occur naturally in the muscle.Future investigations should determine the extent to which these modifications occur in living muscle, early post-mortem muscle and postrigor meat.

Oxidation of calpains by reactive oxygen species
It is known that calpain-1 active sites can be inactivated by reactive oxygen species, such as hydrogen peroxide (Lametsch et al. 2008).Carlin et al. (2006) showed that incubating pure calpain-1 and calpain-2 with hydrogen peroxide slowed the desmin proteolysis rate.Oxidation and inhibition of calpain-1 with hydrogen peroxide were reversible, and a noteworthy modification was an intramolecular disulphide bond involving the act site cysteine (Lametsch et al. 2008).Calpastatin inhibits calpains, but oxidative environments promote calpain activation at pH levels consistent with post-mortem meat (Carlin et al. 2006).These observations illustrate the complicated nature of calpain function as it is affected by reactive oxygen species.

Nitrosylation of calpains
Nitric oxide is a key signalling molecule in many cell types, including muscle (Tengan et al. 2012).NO has been implicated in several cellular processes and is a source of reactive nitrogen species.It reacts with protein sulfhydryls to form S-nitrosothiols.The extent of nitrosylation of meat proteins varies across muscles, which may be linked to observed differences in tenderness (Warner et al. 2005;Huff-Lonergan et al. 2010;Li et al. 2018).Nitrosylation changes alter protein function, structure, and activity in vivo.Proteins influenced by nitrosylation include peroxiredoxin, haemoglobin, ryanodine receptor, and Hif-1a (Foster et al. 2009).Incubation of calpain with nitric oxide donor S-nitroso glutathione (GSNO) resulted in nitrosylation of cysteines at sites 49, 351, 384, and 592 in the catalytic subunit of calpain-1 decreased calpain activity against troponin-T and desmin (Liu et al. 2018).Liu et al. (2019), demonstrated that incubating calpain-1 with a nitric oxide donor (NOR-3) increased the nitrosylation of porcine calpain-1.Incubation with NOR-3 also reduced calpain-1 autolysis and activity against porcine myofibrils in a synergistic fashion with calpastatin.Nitric oxide synthase can be inhibited by Nx-nitro-Larginine methyl ester hydrochloride (L-NAME).Incubation of bovine semimembranosus with L-NAME decreased S-nitrosylation of protein, and incubation with GSNO increased protein nitrosylation (Hou et al. 2020).Calpain-1 autolysis and myofibrillar protein degradation were accelerated when nitrosylation was inhibited with L-NAME.Therefore, nitrosylation events are important factors that regulate calpain activity and protein degradation.What is not yet determined are the specific sites of nitrosylation that influence calpain activity.Future studies must determine the specific nitrosylation sites influencing activity and interaction with calpastatin and potential substrates.