| The meat industry is the largest food industry and the raw meat accounts for around70% of the meat production and consumption in China.However,the quality of raw meat in China is still unstable,especially the problem of high incidence of abnormal meat.Dark cutting(DC)beef is a typical representative of abnormal meat which has high ultimate pH(pHu),dark,firm and dry characteristics.The incidence rate of DC beef in China is as high as 10%,which brings huge economic losses to the meat industry.The huge economic loss is also a difficult issue that restricts the development of the beef industry.DC beef has high pHu(pHu ≥ 6.00),due to insufficient post-mortem anaerobic glycolysis.It has been proven that pre-slaughter stresses have a significant influence on the development of high pHu beef,but few studies have focused on the effect of protein S-nitrosylation on the quality of high pHu beef.A large amount of reactive oxygen and nitrogen species would produce during post-mortem aging.Among which nitrogen species nitric oxide(NO)would modify protein cysteine to form protein S-nitrosylation.Protein S-nitrosylation is a typical redox-dependent post-translational modification of proteins,which regulates the structure,activity and function of proteins through post-translational modifications,thereby regulating the physiological metabolic function of muscle during post-mortem aging and eventually affecting the quality of beef.In the early studies of our laboratory,we mainly carried out systematic research on the effect of protein S-nitrosylation on the quality of fresh pork and its mechanism.However,the influence of protein S-nitrosylation on the quality of fresh beef,especially the research on the formation mechanism of abnormal beef,has not been reported.Therefore,this study firstly identified the differential S-nitrosylated protein and cysteine sites of high pHu and intermediate pHu beef by proteomics,to compare the structures of differential S-nitrosylated proteins and cysteines’ function.Based on the identification results,the focus was to verify and explain the mechanism of protein S-nitrosylation on the formation of high pHu beef from protein characteristics and glycolytic pathways.The specific research content and results are as follows:1.The comparison of meat quality and protein S-nitrosylation degree between high pHu and intermediate pHu beefThe total protein S-nitrosylation degree between high pHu and intermediate pHu beef was compared in this chapter.Longissimus thoracis(LT)muscles(12th to 13 th rib)were transected from the left side of the cattle carcasses within 45 min post-mortem for pH value.Based on the pHu value,the LT muscles were categorized into intermediate pHu beef(5.40< pHu < 5.80,n = 6),and high pHu beef(pHu ≥ 6.00,n = 6)for meat quality measurements and biochemical analysis.The results showed that the color,the purge loss,the cooking loss and the shearing force at 24 h post-mortem of high pHu beef were significantly lower than intermediate pHu beef(P < 0.05).High pHu beef had higher NO content and NOS activity(P < 0.05),and the expression level of neural NOS(n NOS)was significantly higher than that of intermediate pHu beef(P < 0.05).In addition,it was observed by SDS-PAGE that the number and intensity of the main protein bands of the two groups of samples before iodo-tandem mass tags(iodoTMT)labeling were basically the similar,which ensured that the protein content of both groups remained the similar before the S-nitrosylation identification process.Western blot analysis revealed that the band quantities of total S-nitrosylated proteins in high pHu beef increased significantly compared with intermediate pHu beef,and the molecular weight was mainly concentrated between 15-55 k Da.The above results indicated that total protein S-nitrosylation degree was significantly different between high pHu and intermediate pHu beef.The above results showed that the quality and the degree of total protein S-nitrosylation of intermediate pHu beef and high pHu beef were different.The band intensity of S-nitrosylated protein of high pHu beef was higher than that of intermediate pHu beef,and the different proteins had different S-nitrosylation intensity providing basis for the subsequent research on the S-nitrosylated proteins and cysteine sites,as well as the differences in the degree of S-nitrosylation of specific proteins.2.Characteristics and identification of S-nitrosylated proteins and cysteines sites in high pHu and intermediate pHu beefThe LT muscles of high pHu and intermediate pHu beef were analyzed and quantified using isobaric iodoTMT126-129 switch assay to identify the S-nitrosylated proteins and cysteine sites in this chapter.In addition,combined with bioinformatics methods,based on the standard of fold change > 1.2 and coefficient of variation < 0.1,the S-nitrosylated proteins and the cysteine sites were determined through Uni Prot Bovine,and the characteristics were analyzed.The results showed that a total of 856 S-nitrosylated sites from 257 proteins were identified from high pHu and intermediate pHu beef.Among them,there were 131 modification sites up-regulated,whereas 31 modification sites were down-regulated in high pHu beef.The molecular weight and the isoelectric point of S-nitrosylated proteins were distributed in a wide range.The primary structural motif revealed the glycine(G)positioned at-3,-6 and +6 through the S-nitrosocysteine.The different S-nitrosylated proteins in intermediate pHu beef and high pHu beef mainly included energy metabolizing enzyme,oxidoreductases,calcium regulation and mitochondrial dysfunction proteins.Through the analysis of protein interaction and KEGG pathway,the clusters with the closest interaction of S-nitrosylated proteins were glycolytic enzyme cluster and redox protein cluster,while the glycolysis process was a typical metabolic pathway involving S-nitrosylated proteins.The above results indicated that a large amount of cysteine and protein in postmortem beef were modified by S-nitrosylation,and the protein S-nitrosylation level of high pHu beef was significantly higher than that of intermediate pHu beef.The different S-nitrosylated proteins could be involved in energy metabolism,calcium homeostasis and mitochondrial dysfunction in postmortem,which may be involved in regulating meat quality during postmortem aging.3.Study on the effect of protein S-nitrosylation on glycolytic enzymes in beefBased on the previous chapter,the closest interaction clusters were redox protein clusters and glycolytic enzyme clusters,while the glycolysis process was a typical metabolic pathway involving S-nitrosylated proteins.In this chapter,the contents of glycogen and lactic acid were determined.The effects of protein S-nitrosylation on the formation of high pHu beef were investigated by comparing the expression,activity and degree of S-nitrosylation of glycogen phosphorylase(GP),triose phosphate isomerase(TPI)and β-enolase(ENO3),which were three highly differentiated glycolytic enzymes related to meat quality.The glycogen content and the lactic acid content of high pHu beef were significantly lower than intermediate pHu beef at 24 h postmortem(P < 0.05).There was no significant difference in the expression levels of GP,ENO3 and TPI between the two groups(P > 0.05).Compared with intermediate pHu beef,high pHu beef had lower activities and higher S-nitrosylation levels of GP,ENO3 and TPI(P < 0.05).The above results indicated that S-nitrosylated proteins were widely distributed in the muscle,to regulate the activity or function of glycolytic enzymes and participate in the metabolic process of beef postmortem.The higher S-nitrosylation levels of GP,ENO3 and TPI correspond with lower glycolytic enzymes activities.Therefore,protein S-nitrosylation could regulate postmortem glycolysis and meat quality by modulating enzyme activities. |
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