Analysis Of Phosphoproteome Of Ovine Muscle With Different Tenderness

The objective of this study was to investigate the effects of protein phosphorylation on ovine muscle tenderness. Forty crossbred sheep were seleted and the muscle longissimus dorsi were used. The WBSF and MFI were measured to divide the animals into tough and tender group. The global protein phosphorylation level of sarcoplasmic proteins and myofibrillar poteins, protein phosphorylation level of some important sarcoplasmic proteins and myofibrillar poteins were studied. The differential phosphoproteins were identified by two-dimensional electrophoresis. The effects of protein phosphorylation on myofibrillar potein functions were validated by changing the protein phosphorylation level of ovine muscle. The detailed results are list as follows:(1) The changes of sarcoplasmic proteins phosphorylation levels in postmortem ovine muscle with different tenderness were investigated by a combination of SDS-PAGE coupled with Pro Q Diamond-SYPRO Ruby staining. The results indicated that the phosphorylation levels of sarcoplasmic proteins were different at different tenderness group and different postmortem time. The highest protein phosphorylation level of tough group was at 4 h postmortem and the highest protein phosphorylation level of tenderh group was at 12 h postmortem. The phosphorylation levels of sarcoplasmic proteins were increased and then decreased. The global phosphorylation level of tough group was significantly higher than that of tender group at 0.5 h, 1 h and 4 h postmortem(P<0.05).(2) The changes of myofibrillar proteins phosphorylation levels in postmortem ovine muscle with different tenderness were investigated. The results indicated that the phosphorylation levels of myofibrillar proteins were affected by tenderness and postmortem time. The global phosphorylation level of tough group was significantly higher than tender group at 4 h, 12 h and 24 h postmortem(P<0.05). Protein identification revealed that most of the phosphoproteins were proteins with sarcomeric function and the others were involved in glycometabolism, stress response, etc. The ACTB2, TPM1, MYLC2, GP, ENO, LDHA were the main proteins identified in the highly phosphorylated bands.(3) The differential phosphoproteins were identified by two-dimensional electrophoresis. There were 5, 8 and 9 differential phosphoproteins investigated at 0.5 h, 4 h and 24 h postmortem respectively. The differential phosphoproteins of sarcoplasm were GP, GAPDH and PK etc. The GP has 4 isozymes and GAPDH has 2 isozymes and they all related with glycolysis. The differential phosphoproteins of myofibril were MYBPH, ACTN3 and TPM1 and they all related to muscle contraction. Therefore, the protein phosphorylation may affect tenderness through glycolysis and muscle contraction.(4) Myofirillar protein functions after modifying muscle protein phosphorylation level by protein kinase were investigated. The activity of protein kinases in PMA group(PKC activated group) were higher than control group after 1 h, 2 h and 4 h incubation(P<0.05). The activity of protein kinases in Forskolin group(PKA activated group) were higher than control group after 1 h and 4 h incubation(P<0.05). The highest activity of protein kinases was appeared after 1 h incubation. PKA and PKC phosphorylated TTN, MYBPC, TPM, MYLC2 and a protein of 25 k Da. Phosphorylation of some myofibrillar proteins resulted in reduced degradation of these proteins by the calpains, shortened of sarcomere length and muscle contraction.