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BACKGROUND The abnormal ultimate pH (pHu ) in postmortem muscles affect the meat quality and results in substantial economic losses. Dark, firm, and dry (DFD) meat linked with the higher postmortem pHu values and exhibited many quality issues such as dark color, tough texture and shorter shelf life. This research aimed to investigate the effect of protein phosphorylation on variations in beef pHu in order to explore the possible mechanisms underlying DFD meat formation. RESULTS Glycogen and lactate contents were higher, while L* and a* were lower in high pHu beef. Shear force was higher in intermediate pHu group. Global phosphorylation of sarcoplasmic proteins was higher in low pHu samples on day 1 and of myofibrillar proteins was higher in intermediate pHu meat on days 1 and 2 postmortem. Sarcoplasmic protein bands with different phosphorylation levels were identified as containing some glycometabolism and stress response proteins and phosphorylated myofibrillar protein bands were identified sarcomeric and metabolic proteins. CONCLUSIONS Phosphorylation of multiple proteins of glycolytic pathway and contractile machinery may play critical roles in development of DFD beef. © 2021 Society of Chemical Industry.

Phosphorylation post-translational modification plays an important role in postmortem muscle quality traits. Adenosine triphosphate (ATP) is an energy source and a key substrate of phosphorylation which provides the phosphatase groups to proteins in the presence of protein kinases. However, in postmortem muscle, the effects of ATP content on phosphorylation are poorly studied. The study investigated the effect of ATP on protein phosphorylation and degradation in postmortem ovine muscle. The ground muscle with/without additional ATP were treated/control groups and stored at 25 and 4°C, respectively. The ATP content led to different changes of pH value between the ATP-treated and control groups. The phosphorylation level of myofibrillar proteins was higher (P

Meat quality plays an important role in the purchase decision of consumers, affecting producers and retailers. The formation mechanisms determining meat quality are intricate, as several endogenous and exogenous factors contribute during antemortem and postmortem periods. Abundant research has been performed on meat quality; however, unexpected variation in meat quality remains an issue in the meat industry. Protein posttranslational modifications (PTMs) regulate structures and functions of proteins in living tissues, and recent reports confirmed their importance in meat quality. The objective of this review was to provide a summary of the research on the effects of PTMs on meat quality. The effects of four common PTMs, namely, protein phosphorylation, acetylation, S-nitrosylation, and ubiquitination, on meat quality were discussed, with emphasis on the effects of protein phosphorylation on meat tenderness, color, and water holding capacity. The mechanisms and factors that may affect the function of protein phosphorylation are also discussed. The current research confirms that meat quality traits are regulated by multiple PTMs. Cross talk between different PTMs and interactions of PTMs with postmortem biochemical processes need to be explored to improve our understanding on factors affecting meat quality.

Water holding capacity and its corollary, drip loss, are important meat quality indicators. The high drip loss muscle had lower pH, higher brightness, higher shear force value, and shorter sarcomere length than the low drip loss muscle. The protein phosphorylation level of different drip loss muscles was detected via the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in this study. The results showed that the phosphorylation level of sarcoplasmic and myofibrillar in the high drip loss group was higher than that in the low drip loss group. Combined with the gray value analysis, the bands with higher phosphorylation levels of sarcoplasmic proteins were mainly enzymes involved in glycolysis which could affect the pH; while the bands with higher phosphorylation levels of myofibrillar proteins were mainly cytoskeletal proteins and contractile proteins. It can be concluded that the low pH and increased muscle contraction had a major influence on muscle drip loss.

With the advancement of technical approaches, signaling pathways have been continuously discovered in numerous biological processes. Majority of these signaling pathways include the protein phosphorylation, which is a main post-translational modification affecting the structure and function of proteins within the cell. Consequently, determining the locations and extent of the protein phosphorylation of any specific protein or set of proteins, either qualitatively or quantitatively, has become a routine and extremely vital step in many fields of life sciences, and recently in meat science as well. However, when choosing the method to detect protein phosphorylation, it can be different depending on several aspects, particularly under consideration of the availability of the related reagents and equipment. Although different methods have their own advantages, but this is also a matter of fact that every method is associated with several drawbacks such as poor specificity, low sensitivity, safety issues, or intensive labor. In this chapter, we outlined the routine methods used to detect the protein phosphorylation and discussed their advantages and disadvantages, especially focusing on the usage of gel and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques to identify phosphorylated proteins in meat science field.

Glycolysis, the core metabolic pathway in muscle, is very significant to the ultimate meat quality. The objective of this chapter was to confirm the involvement of protein phosphorylation on postmortem glycolysis and the possible regulatory mechanism from the perspective of enzyme activity. The global phosphorylation level of sarcoplasmic protein increased early postmortem and then decreased afterwards in muscle. Protein bands with significantly different phosphorylation level were identified as glycometabolism related enzymes. By applying quantitative proteomic tools (isobaric Tags for Relative and Absolute Quantitation, iTRAQ) and bioinformatics analysis, 24 phosphoproteins clustered in glycolysis and muscle contraction were identified to be glycolytic rate related proteins, and phosphorylation of pyruvate kinase at Thr 157 was negatively correlated with glycolytic rate. Furthermore, the animal muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor. Protein phosphorylation was positively correlated with the activity of glycogen phosphorylase, pyruvate kinase, and phosphofructokinase. In conclusion, protein phosphorylation played a role in postmortem glycolysis through the regulation of enzyme activity in muscle. Protein phosphorylation was related to postmortem glycolysis and glycolytic enzymes, and the phosphorylation level of glycolytic enzymes may influence its activities, further regulating postmortem glycolysis.

As the most important pigment for meat color, myoglobin mainly exists in sarcoplasm, and meat color is determined by its absolute content as well as the relative content of three types of myoglobin (oxymyoglobin, deoxymyoglobin, and metmyoglobin). Previous studies have shown that protein phosphorylation may negatively regulate the stability of meat color through the regulation of glycolysis pathway and myoglobin. The purpose of this research was to study the influence of phosphorylation on myoglobin stability, in order to provide a theoretical basis for improving the stability of meat color through regulating phosphorylation level. Sodium hydrosulfite was used to reduce myoglobin from skeletal muscle, and later was removed by ultrafiltration. Then alkaline phosphatase (AP) was added to in vitro incubation system to catalyze the dephosphorylation of myoglobin. The changes in myoglobin phosphorylation level were measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Pro-Q Diamond and Ruby phosphoprotein gel staining. We also kept a record of the changes in pH and used ultraviolet spectrophotometer to measure the relative content of three myoglobin redox forms. The ultraviolet spectrophotometer of myoglobin was determined by circular dichroism (CD) spectroscopy. Myoglobin phosphorylation may lead to changes in its secondary structure, therefore reducing myoglobin stability and increasing its autoxidation rate, which further accelerated the accumulation of metmyoglobin. As a result, meat color was found to have a low stability, and this might be one of the explanations that protein phosphorylation negatively regulated meat color stability.

Tenderness is one of the most important quality attributes especially for beef and lamb. As protein phosphorylation and dephosphorylation regulate glycolysis, muscle contraction, and turnover of proteins within living cells, it may contribute to the conversion of muscle to meat. The protein phosphorylation level (P/T ratio) of the tender group increased from 0.5 to 12 h postmortem and then decreased. The P/T ratio of tough group increased during 24 h postmortem, with higher increase rate from 0.5 to 4 h postmortem than from 4 to 24 h postmortem. The global phosphorylation level of tough meat was higher than tender meat at 4, 12, and 24 h postmortem. Protein identification revealed that most of the phosphoproteins were proteins with sarcomeric function; the others were involved in glycometabolism, stress response, etc. The phosphorylation levels of myofibrillar proteins, e.g. myosin light chain 2 and actin, were significantly different among groups of different tenderness and at different postmortem time points. Protein phosphorylation may influence meat rigor mortis through contractile machinery and glycolysis, which in turn affected meat tenderness.

Salting is one of the most ancient preservation methods for food, which is widely used to preserve meat and improve the quality of meat products, such as water holding capacity (WHC), texture, tenderness, flavor, taste, and so on. Protein phosphorylation is one of the most ubiquitous post-translational modifications (PTMs). Reversible protein phosphorylation plays an important role in protein structure, function, signaling, and regulation. As one of the most essential processes for meat products, the effects of salting on muscle protein phosphorylation are unclear. Thus, we investigated the phosphorylation of myofibrillar and sarcoplasmic proteins in response to salt content and salting time. We discovered that salting showed significant effect on myofibrillar protein phosphorylation. Four categories of phosphorylated protein were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), involved in stress response (heat shock protein), glycometabolism (glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase), oxidation or reduction (superoxide dismutase), and others (myoglobin). The phosphorylation level of the proteins mentioned above was affected by salting. Thus, salting might influence meat quality through protein phosphorylation, which regulated protein degradation and glycolysis. Furthermore, ten different phosphoproteins (>1.5 fold) induced by salting were identified by LC-MS/MS and the UniProt database, most of which were involved in glycometabolism, protein function, and protein degradation. It was concluded that salting might influence meat quality through protein phosphorylation, which regulated glycolysis metabolism, protein function, and degradation.

μ-Calpain is the key enzyme in postmortem meat tenderization. Studies have shown that μ-calpain can be phosphorylated and phosphorylation influences the activity of μ-calpain. However, whether μ-calpain can be phosphorylated in postmortem muscles and the possible relative influencing mechanism remains unknown. The longissimus lumborum (LL) muscles were used in this research. The relationship between phosphorylation level of sarcoplasmic proteins, μ-calpain activity, and calpastatin degradation in mutton with different tenderness was analyzed. Alkaline phosphatase and phosphatase inhibitor were used to modulate the phosphorylation level of sarcoplasmic proteins, and the effect of phosphorylation of sarcoplasmic proteins on μ-calpain activity was studied. The phosphorylation level of μ-calpain was regulated in vitro, and the influence of dephosphorylation and phosphorylation of μ-calpain on μ-calpain activity was investigated. The changes in the secondary structure of μ-calpain were studied and the phosphorylation sites of μ-calpain were identified to illustrate the directly regulatory mechanism of μ-calpain activity by phosphorylation. The effects of calpastatin on phosphorylated μ-calpain and the effects of phosphorylated calpastatin on μ-calpain were also analyzed. The study of the effects of phosphorylation on the interaction between calpastatin and μ-calpain clarified the indirectly regulatory mechanism of phosphorylation on μ-calpain activity.

Myofibrillar proteins degradation contributes to meat tenderization during postmortem. Protein phosphorylation has been revealed to be associated with meat tenderness in recent years. This chapter aimed to investigate the effect of myofibrillar protein phosphorylation on the degradation susceptibility by μ-calpain. Protein kinase A (PKA) and alkaline phosphatase (AP) were added to myofibrillar proteins, extracted from ovine longissimus lumborum (LL) muscles, to modify the phosphorylation level for 30 min at 30 °C. The samples were then incubated with μ-calpain at 4 °C at different Ca2+ concentrations. The results showed that more degradation products of myosin heavy chain, actin, titin, desmin, and troponin T were detected with the increase of Ca2+ concentration. The protein degradation was higher in AP treated group compared to the control group. This research indicated that phosphorylation prevented proteolytic susceptibility of myofibrillar proteins to degradation by μ-calpain. Protein phosphorylation made a major contribution in meat tenderization during postmortem.

Extensive studies have revealed that protein phosphorylation plays an important role in postmortem meat quality, such as tenderness, color, and water holding capacity. During storage of meat, the storage temperature is one of the most important factors that determine meat quality. High temperature promotes meat spoilage by accelerating biochemical reactions and microbial growth. However, whether storage temperature affects the development of meat quality through phosphorylation is not well understood. In order to investigate it clearly, the postmortem ovine muscle samples were stored at 25 °C, 15 °C, 4 °C, and −1.5 °C from 0.75 h to 21 days. The correlation between temperature, pH, adenosine triphosphate (ATP) content, and phosphorylation levels was analyzed, and then differential phosphorylated proteins were identified by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gene ontology analysis. Myosin binding protein C, troponin T3, actinin, myosin light chain, heat shock protein 90, glucose-6-phosphate, pyruvate kinase, enolase, fructose-bisphosphate aldolase, L-lactate dehydrogenase, etc. were phosphorylated in postmortem ovine muscles. Moreover, ATP content was a vital factor which had the strongest correlation with the phosphorylation level of individual protein band. In order to prove whether ATP content mainly affected phosphorylation level, ATP was added to ground ovine muscle in vitro, which showed that ATP improved the phosphorylation level of myofibrillar proteins. The phosphorylation reaction was reversible. However, ATP was unnecessary for dephosphorylation. Whether the particular temperature and pH (5.2, 5.8, 6.4) had effect on the dephosphorylation of myofibrillar protein through alkaline phosphatase are need to be studied. The result showed that −1.5 °C and pH 5.2 restrained the activity of alkaline phosphatase and decreased phosphorylation level. Phosphorylation was an essential post-translational modification and more insights were needed for the complicated formation in postmortem muscle.

The relationship between meat color stability and protein phosphorylation was studied. By adding phosphatase and protein kinase inhibitors into sarcoplasmic proteins from minced ovine longissimus thoracis et lumborum (LTL) muscle, we found out that protein phosphorylation had a significant relationship with lactate accumulation, as well as pH decline rate and extent. Also, meat color stability had a negative correlation with sarcoplasmic protein phosphorylation level. Therefore, how protein phosphorylation regulated glycolysis and myoglobin redox forms, and furthermore meat color stability was investigated in the second step. Results showed that phosphorylation differences existed in some individual protein bands, but not in whole sarcoplasmic proteins, from muscle groups with different meat color stability. Glycolytic enzymes turned out to be the most color stability related proteins, and myoglobin phosphorylation level was negatively related to meat color stability, which meant protein phosphorylation might regulate meat color stability via glycolysis and myoglobin redox stability. At the third step, the protein phosphorylation level of muscles with different color stability was examined with quantitative analysis. We successfully identified 3412 phosphopeptides from 1070 phosphoproteins, and among which 243 had a significant correlation between phosphorylation level and meat color stability. With further study into these phosphopeptides through informatics analysis, 27 of them were highly related to meat color stability, and glycolytic enzymes were the biggest group. Moreover, there was a negative correlation between the Ser133 phosphorylation level in myoglobin and meat color stability. In conclusion, our results revealed that protein phosphorylation may regulate meat color stability through glycolytic enzymes and myoglobin redox forms, and Ser133 played a key role in the process.

The aim of this study was to assess the effects of the phosphorylation levels of glycogen phosphorylase on its activity in mutton sarcoplasmic protein samples during incubation at 4 °C. Samples of sarcoplasmic proteins from mutton longissimus thoracis muscles were prepared and separated into three treatment groups to obtain glycogen phosphorylase with different phosphorylation levels, which were (1) treated with protein kinase A, (2) treated with alkaline phosphatase, and (3) left untreated (control). Glycogen phosphorylase phosphorylation levels and activity as well as the levels of related endogenous substances were assessed. The results showed that phosphorylation of glycogen phosphorylase in mutton promoted its activity during incubation at 4 °C. The activity of glycogen phosphorylase was also influenced by other factors (glycogen, glucose, glucose 6-phosphate, ATP, etc.) in vitro. The combined effects of phosphorylation and endogenous substances on glycogen phosphorylase activity varied at different incubation times.

The relationship between glycogen phosphorylase activity and phosphorylation levels in the longissimus thoracis muscle post-mortem was studied. Sixty lamb samples were collected at 0.5 h, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h post-mortem and divided into three groups (n = 6) with different glycolytic rates (fast, intermediate, and slow) according to the pH at 6 h post-mortem. The phosphorylation level and activity and expression of glycogen phosphorylase were determined. The results showed that the phosphorylation level and activity of glycogen phosphorylase in the slow pH decline group was lower than that in the fast pH decline group during 24 h post-mortem (P

Phosphorylation of myofibrillar proteins can regulate muscle contraction and thus affect actomyosin dissociation. To explore the mechanism by which myofibrillar protein phosphorylation and phosphorylation site affects actomyosin dissociation, mass spectrometry-based quantitative phosphoproteomics was used to analyze ovine longissimus thoracis muscles with different actomyosin dissociation. A total of 2073 phosphorylated peptides from 826 phosphoproteins were identified among the myofibrillar proteins of muscles with different actomyosin dissociation, and 176 phosphorylated peptides from 114 phosphoproteins were significantly different (P high and low group of actomyosin dissociation . Phosphorylation of myosin heavy chain 1 at Ser1896, myosin heavy chain 7 at Thr1019 and Tyr1307 , and titin at Ser265, Ser6085, Ser21930, and Ser26576 can inhibit actomyosin dissociation, whereas phosphorylation of tropomyosin 2 at Ser86, Thr252, and Ser279 contribute to actomyosin dissociation. The phosphorylation sites associated with actomyosin dissociation were mostly serine residues, and the phosphorylation status of myosin regulatory light chain at Ser15 was negatively correlated with actomyosin dissociation. In conclusion, the phosphorylation of some myofibrillar proteins and myosin at specific serine residues may be crucial for actomyosin dissociation.