The Effect Of Protein Phosphorylation On The Ovine Meat Quality

Muscle protein phosphorylation modification is an important approach to regulate the meat quality after slaughter.The mutton with the local characteristic from Hengshan,north of shaanxi province,is the national product of geopgraphical indication,and constomers pay more attention to its quality.This paper investigated the effect of peotein phosphorylation on the Hengshan white-cashmere goat meat quality with the aim to decipher the regulation mechanism of protein phosphorylation for high quality mutton and low quality mutton,respectively.This study can provide the theoretical foundation for high quality Hengshan mutton production.The longissimus dorsi and breast&lap of white-cashmere goats which were respectively regarded as high quality meat(HQ)and low quality meat(LQ)were used as the raw material for this study,The differences of these two kinds of meat were determined according to meat quality attributes(pH value,shear force,color,protein solubility and water-holding capacity)and myofibrillar protein degradation within 48h postmortem.The phosphopeptides of two groups was used for LC-MS/MS analysis.The bioinformation analysis of identified phosphoprotein was performed by GO and KEGG strategy to explore the regulation mechanism of protein phosphorylation on meat quality.This study provides basis for the regulation of protein phosphorylation on meat quality by combination of phosphoproteins identification,quantitative analysis and bio information of phosphoproteins.The major results are listed as follows:(1)Mutton quality changes within 48h postmortem.The meat quality attributes such pH value,shear force,color,protein solubility and water-holding capacity were determined to investigate the change of meat quality after slaughter.The results showed that meat pH significantly declined with the prolonged postmortem time while the rate and amplitude of pH decline for HQ were lager than that for LQ(p<0.05).Mutton shear force without changed within 8h postmortem,but it showed significantly decreased after 8hpostmortem and the meat shear force in HQ was lower than LQ meat(P<0.05),and which sharply decreased at 12h for both HQ and LQ.Meanwhile,meat color analysis found that the increasing of L*and decreasing of a*for HQ were more remarkable than LQ(p<0.05)during postmortem,while b*was not obviously influenced by postmortem time(p>0.05).Although there was significant difference between HQ and LQ in the sarcoplasmic protein solubility and the myofibrillar protein solubility(p<0.05),the discipline of the changing was same.The sarcoplasmic protein solubility constantly increased while the myofibrillar protein solubility decreased at first and then increased.The HQ myofibrillar protein solubility changed more intense than LQ(p<0.05).The protein electrophoresis showed that the myofibrillar protein degradation degree of HQ was higher than LQ.Above all,there are significant differences in meat quality attributes between HQ and LQ.(2)The results for phosphopeptides quantitation and identification by LC-MS/MS.The phosphopeptides were analyzed by LC-MS/MS.In total 2125 phosphopeptides were obtained by searching for NCBI_Ovis database,and these phosphopep tides were assigned to 750 p ho sp hop rote ins.The phosphoserines,p ho sp ho threonines and phosp ho tyrosines respectively account for 82%,12%and 6%of the total phosphorylation residues identified.After quantitative analysis,the phosphorylation level of 968 unique phosphopeptides were found to be significantly changed,in which the phosphorylation level of 387 phosphopeptides in HQ group was up-regulated,while 581 phosphopeptides in LQ group with their phosphorylation level up-regulated.The 968 phosphopeptides were assigned to 433 phosphoproteins,and the proteins with significant phosphorylation level difference between HQ and LQ were Phosp ho frutokinase(PFK),pyruvate kinase(PK),Glyc era ldehyde-3-phosp hate dehydro genase(GAP DH),triosephosphate isomerase(TPI1),Fructose-bisphosphate aldolase A(ALDOA),myosin(MYH1,MYH2,MYH6),tropomyosin(TPM),Titin,Myosin regulatory light chain 2(MYL2),troponin I,Sarcoplasmic/endoplasmic reticulum calcium ATPase 2(ATP2A2),Heat shock protein(HSP27 and HSP70-1A/B).(3)The exploration for regulation mechanism of protein phosphorylation on ovine meat quality.GO and KEGG enrichment strategy was applied to investigate the molecular function and related biochemical process of total 750 phosphoproteins for deciphering the effects of protein phosphorylation on mutton quality.Results showed that catalytic activity,molecular function regulator and binding were found to be the major protein function.82.35%phosphoproteins,participated in the biological process of metabolism,signaling,response to stimulus and biological regulation,were involved in the three above functions.The phosphoproteins involved in 23 pathyways which was enriched by KEGG.About 71.23%phosphoproteins were significantly enriched in glycolysis,glycogen metabolism,Ca2+ signaling pathway,muscle contraction and stress response.Moreover,the protein-protein interaction network for the phosphoproteins with significant phosphorylation level difference in HQ group and LQ group showed that the enzymes involved in glycolysis was the core of the protein-protein interaction network of HQ group,while protein-protein interaction network of LQ group showed that the proteins related to muscle contraction was the core of the network.Therefore,phosphorylation modification can regulate the activity of glycolytic enzymes thereby affecting the rate of meat pH decline and ATP producing.And phosphorylation of PFK,PK,GAPDH,TPI1,ALDOA may affect meat glycolysis rate.Phosphoproteins participated in Ca2+ signaling pathway and muscle contraction play a role in regulating the releases Ca2+ from sarcoplasmic reticulum and the formation of actomyosin,respectively.These processes lead to the irreversible muscle contraction and have a negative effect on meat tenderness.Meanwhile,AIP2A2,TPM,Titin and MYL2 phosphorylation affect the two processes.The proteolytic degradation of structural proteins is an important factor in meat quality changes.Proteolytic degradation induces phosphorylation of stress resistance proteins to improve their capacity of protection structural protein from degradation Thus phosphorylation of troponin I,heat shock proteins(HSP27 and HSP70-1A/B)may have positive effect on protein degradation resistant.Furthermore,muscle pH decline induce protein denaturation and the changes of its solubility.As proteolysis degradation of myofibrillar proteins would cause water loss of muscle cell.In addition,there was more intracellular water flowing out and accumulating on the muscle surface which led to that the increasing of L*and decreasing of a*during postmortern.In conclusion,protein phosphorylation has an effect on meat quality through the regulation of proteins involved in glucose metabolism,muscle contraction as well as proteolytic degradation of proteins.