A Combined Differential Proteome and Transcriptome Profiling of Fast- and Slow-Twitch Skeletal Muscle in Pigs

Abstract

Skeletal muscle fiber types can contribute in part to affecting pork quality parameters. Biceps femoris (Bf) (fast muscle or white muscle) and Soleus (Sol) (slow muscle or red muscle) are two typical skeletal muscles characterized by obvious muscle fiber type differences in pigs. However, the critical proteins and potential regulatory mechanisms regulating porcine skeletal muscle fibers have yet to be clearly defined. In this study, the isobaric Tag for Relative and Absolute Quantification (iTRAQ)-based proteome was used to identify the key proteins affecting the skeletal muscle fiber types with Bf and Sol, by integrating the previous transcriptome data, while function enrichment analysis and a protein-protein interaction (PPI) network were utilized to explore the potential regulatory mechanisms of skeletal muscle fibers. A total of 126 differentially abundant proteins (DAPs) between the Bf and Sol were identified, and 12 genes were found to be overlapping between differentially expressed genes (DEGs) and DAPs, which are the critical proteins regulating the formation of skeletal muscle fibers. Functional enrichment and PPI analysis showed that the DAPs were mainly involved in the skeletal-muscle-associated structural proteins, mitochondria and energy metabolism, tricarboxylic acid cycle, fatty acid metabolism, and kinase activity, suggesting that PPI networks including DAPs are the main regulatory network affecting muscle fiber formation. Overall, these data provide valuable information for understanding the molecular mechanism underlying the formation and conversion of muscle fiber types, and provide potential markers for the evaluation of meat quality.

Keywords: meat quality; muscle fiber; pig; proteome; transcriptome.

Figure 1

GO and KOG annotation and classification of all identified proteins. (A) GO classification of all the proteins identified. x axis represents the GO terms in three types of GO ontology, and the y axis represents the gene number. (B) KOG classification of all proteins identified. x axis refers to function class; y axis represents the gene number.

Figure 2

Principal component analysis (PCA). Based on the expression of the identified proteins, PCA was carried out to visualize the differences between Bf and Sol samples.

Figure 3

Identification of DAPs between fast-twitch and slow-twitch muscles. A volcano plot was drawn to show the DAPs between Bf and Sol. The green dots indicate significantly downregulated proteins (p < 0.01 and fold change <0.83), the blue dots indicate significantly downregulated proteins (p < 0.05 and fold change <0.83), the yellow dots indicate significantly upregulated proteins (p < 0.01 and fold change >1.2), the red dots indicate significantly upregulated proteins (p < 0.05 and fold change >1.2), and the black dots represent proteins with non-significant (p > 0.05 or 0.83 < fold change <1.2) differences in expression.

Figure 4

GO and KEGG pathway enrichment analysis of DAPs. (A) GO enrichment analysis of DAPs. (B) KEGG pathway enrichment of DAPs. The top 20 enrichment GO terms and pathways are shown, and detailed information can be found in Table S5.

Figure 5

Protein–protein interaction (PPI) network of DAPs. (A) PPI network of all the DAPs. (B) PPI network of the DAPs from the top 20 most significantly enriched KEGG pathways. The interaction network was constructed using the web-based search STRING database. Line color indicates the type of interaction evidence: the light blue line indicates the known interactions from the curated databases, the purple line indicates the known interactions that were experimentally determined, the blue line indicates the predicted interactions of co-occurrence genes, and the yellow line indicates the PPI using the text mining method. Solid line represents the PPIs that were experimentally validated, whereas the dotted line represents the PPIs that have not yet been validated.

Figure 6

Integrated analysis of transcriptome and proteome. (A) Overall correlation between mRNA and protein changes displayed using a nine-quadrant diagram. x axis represents log₂ (fold change) of proteins and y axis represents the log₂ (fold change) of mRNAs. The correlation coefficient and p value of the transcriptome and proteome are shown at the top of the graph. Each dot represents a gene and protein. The black dots represent non-differentially expressed proteins and genes; the red dots represent the genes and proteins whose expression trends are consistent or opposite; the green dots represent the DEGs but not DAPs; and the blue dots represent DAPs but not DEGs. (B) Venn plot of DAPs and DEGs. The gene name and Ensemble ID for the 12 overlapped genes are shown in the “Results” section of the main text.