Comparative analysis of muscle phosphoproteome induced by salt curing
Introduction
Salting is commonly used in meat processing to improve the quality of processed meat products like flavour, water holding capacity, tenderness, juiciness, firmness, gelation and appearance (Comaposada et al., 2000, Duranton et al., 2012, Shao et al., 2016). NaCl is the essential salting material used in cured or cooked meat products. It increases the ionic strength within muscle, which causes protein dissociation, denaturation, modification and myofibril swelling (Kim et al., 2015, Shao et al., 2016, Wang et al., 2016).
Protein phosphorylation is the most common post-translational modification (PTM). The majority of muscle proteins can be phosphorylated in vivo. Protein phosphorylation affects protein stability, enzymatic activity and muscle contraction, which may then affect muscle development, carcass rigor mortis and meat quality (Huang et al., 2012, Li et al., 2012). As NaCl influences meat quality by regulating myofibrillar protein denaturation, actomyosin dissociation, myosin hydration and myoglobin redox (Duranton et al., 2012, Kim et al., 2015, Wang et al., 2016), while myosin, actin and myoglobin could be phosphorylated (Chen et al., 2016, Li et al., 2017). Whether NaCl influences protein phosphorylation and the phosphorylation alters meat quality, which should be elucidated by further research.
Many salt-responsive phosphoproteins have been identified in vivo in plants (Hu, Guo, Li, & Ren, 2013). Under NaCl stress, the ionic equilibrium of cells is disturbed, which significantly alters the phosphorylation of ATPases, sodium transporers and aquaporins by AGC (cAMP-dependent, cGMP-dependent and protein kinase C) kinases (Vialaret et al., 2014). In mice, salt regulates the phosphorylation of SPAK and NKCC1 in aorta (Zeniya et al., 2013). Higher salt intake decreases the phosphorylation of WNK-SPAK-NKC1 in the vascular smooth muscle, resulting in disorganized common vascular tone (Zeniya et al., 2013). Our previous study has also revealed that salt curing affect the global phosphorylation of myofibrillar and sarcoplasmic proteins, including heat shock protein, glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, and superoxide dismutase (Zhang et al., 2016), which may be a mechanism by which salt curing improves meat quality. However, the differential phosphorylated proteins were separated by SDS-PAGE, which couldn't identify the whole muscle phosphoproteome induced by salt clearly, and couldn't answer the influence of salt on the phosphorylation and function of muscle proteins appropriately.
In this study, the phosphoproteome in ovine muscle salted with or without 3% NaCl was comparatively studied by 2-D electrophoresis in combination with LC-MS/MS identification of differentially phosphorylated proteins. By analyzing the differential phosphorylate proteins in response to sodium chloride, to confirm the influences of salting on muscle protein phosphorylation, and to reveal a potential new mechanism by which salt alter meat quality.
Section snippets
Sample preparation
Five male (not castrated), 8-month old sheep (large Tailed Han sheep × small Tailed Han sheep) were selected for the experiment. All sheep were grown up in the same feed lot under the same feeding conditions, and slaughtered in a local commercial abattoir according to the industrial practice. The topside muscles were removed immediately from carcasses (weighted from 25 to 30 kg), wrapped with polyethylene film (oxygen permeation rate 10,600 cm3/m2·24 h atm, NuoBang Plastics Co. Ltd. Shanghai, China)
Identification of differentially phosphorylated proteins after salting
After electrophoresis, gels were stained with Pro-Q Diamond for phosphoproteis (P) and with SYPRO Ruby for total proteins (T). The relative phosphorylation levels of proteins were evaluated by ratios of relative protein abundance between control and salted group. As shown in Fig. 1. 13 protein spots were detected to be differentially phosphorylated (ratio of relative protein abundance < 2/3 or > 1.5). The isoelectric points (pI) of the differentially phosphorylated proteins were pH 5–8 and the
Conclusions
In summary, salt curing influences phosphorylation and dephosphorylation of muscle proteins, which accelerates glycolysis metabolism, weaken the protein positive function to meat quality and regulate the muscle contractile to cause the actomyosin dissociation. Probably, phophorylation modification altered by salt curing might be a new mechanism to regulate meat quality, especially the color, tenderness of meat.
Acknowledgments
The authors thank for the financial support from National Natural Science Fund (No.31671879), Special Fund for Agro-scientific Research in the Public Interest (201303082), China Agriculture Research System (CARS-39), Modern Agricultural Talent Support Program-Outstanding Talents and Innovative Team of Agricultural Scientific Research (2016–2020) and National Agricultural Science and Technology Innovation Program (meat science and technology). The authors appreciated to Caoyuanhongbao Industry
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Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, No. 1 Nongda South Rd., Xi Beiwang, Haidian District, Beijing 100193, China.