Mechanisms of Mitochondrial Apoptosis-Mediated Meat Tenderization Based on Quantitative Phosphoproteomic Analysis

Abstract

This study investigates the mechanism of phosphorylation in the regulation of apoptosis-mediated meat tenderization during postmortem aging. The results found that the pork muscle exhibited apoptotic potential at early postmortem (48 h) and showed more tenderness at late postmortem, as evidenced by the increase in mitochondrial membrane permeability (MMP), Ca2+ level, reactive oxygen species (ROS) content, and caspases activity at 0 h to 48 h, and decreases in ATP level at 0 h to 24 h and shear force at 12 h to 120 h (p < 0.05). Phosphoproteomic analysis revealed that phosphorylation regulated apoptosis by modulating ATP and calcium bindings as well as apoptotic signaling, which occurred within early 12 h and mainly occurred at 12 h to 48 h postmortem. Moreover, differential expression of phosphoproteins demonstrated that phosphorylation regulated oxidative stress-induced apoptosis and rigor mortis, thereby promoting the development of meat tenderness. Our results provide insights into the roles of phosphorylation in various physiological processes that affect meat tenderness.

Keywords: apoptosis; muscle; postmortem; proteins; quantitative phosphoproteomic; tenderness.

Figure 1

Changes in apoptosis related factors and tenderness in postmortem pork muscle. (a) mitochondrial membrane permeability (MMP), (b) Ca²⁺ level, (c) ATP level, (d) reactive oxygen species (ROS) content, (e) caspase-9/-3 activity, (f) shear force. Different capital letters represent the difference between aging (p < 0.05).

Figure 2

Identification of differentially expressed proteins in postmortem pork muscle. (a) The schematic overview for the quantitative analysis of pork muscle proteome and phosphoproteome at 0 h to 48 h postmortem. (b) Two-dimensional scatter plot of PCA distribution of protein quantitation. (c) RSD of protein quantitation. (d) Volcano plot of the differentially expressed proteins between 0 h and 12 h postmortem. (e) Volcano plot of the differentially expressed proteins between 12 h and 48 h postmortem. (f) Quantitative analysis of differentially expressed proteins in different sample comparable groups.

Figure 2

Identification of differentially expressed proteins in postmortem pork muscle. (a) The schematic overview for the quantitative analysis of pork muscle proteome and phosphoproteome at 0 h to 48 h postmortem. (b) Two-dimensional scatter plot of PCA distribution of protein quantitation. (c) RSD of protein quantitation. (d) Volcano plot of the differentially expressed proteins between 0 h and 12 h postmortem. (e) Volcano plot of the differentially expressed proteins between 12 h and 48 h postmortem. (f) Quantitative analysis of differentially expressed proteins in different sample comparable groups.

Figure 3

Functional analysis of differentially expressed proteins in postmortem pork muscle. (a,b) Subcellular localizations of differentially expressed proteins at 0–12 h postmortem and 12–48 h postmortem, respectively. (c,d) GO functional enrichment bubble plot of the differentially expressed proteins at 0–12 h postmortem and 12–48 h postmortem, respectively.

Figure 3

Functional analysis of differentially expressed proteins in postmortem pork muscle. (a,b) Subcellular localizations of differentially expressed proteins at 0–12 h postmortem and 12–48 h postmortem, respectively. (c,d) GO functional enrichment bubble plot of the differentially expressed proteins at 0–12 h postmortem and 12–48 h postmortem, respectively.

Figure 4

(a) Mfuzz analysis of differentially expressed protein profiles in postmortem pork muscle. The shade of red and blue represents high expression and low expression, respectively. (b) Protein–protein interaction (PPI) networks of differentially expressed proteins in postmortem pork muscle. The interaction network only presented proteins with connections.

Figure 4

(a) Mfuzz analysis of differentially expressed protein profiles in postmortem pork muscle. The shade of red and blue represents high expression and low expression, respectively. (b) Protein–protein interaction (PPI) networks of differentially expressed proteins in postmortem pork muscle. The interaction network only presented proteins with connections.

Figure 5

Heatmaps of interacting differentially expressed proteins in postmortem pork muscle. The shade of red and blue represents high expression and low expression, respectively.