Isobaric Tagging for Relative and Absolute Protein Quantification (iTRAQ)-Based Quantitative Proteomics Analysis of Differentially Expressed Proteins 1 Week After Spinal Cord Injury in a Rat Model

Abstract

BACKGROUND Spinal cord injury (SCI) is a devastating trauma of the central nervous system (CNS), with high levels of morbidity, disability, and mortality. One week after SCI may be a critical time for treatment. Changes in protein expression have crucial functions in nervous system diseases, although the effects of changes occurring 1 week after SCI on patient outcomes are unclear. MATERIAL AND METHODS Protein expression was examined in a rat contusive SCI model 1 week after SCI. Differentially expressed proteins (DEPs) were identified by isobaric tagging for relative and absolute protein quantification (iTRAQ)-coupled liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomics analysis. Gene Ontology (GO) analysis was performed to identify the biological processes, molecular functions, and cellular component terms of the identified DEPs, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to identify key enriched pathways. Protein-protein interaction (PPI) networks were analyzed to identify the top 10 high-degree core proteins. RESULTS Of the 295 DEPs identified, 204 (69.15%) were upregulated and 91 (30.85%) were downregulated 1 week after injury. The main cellular components, molecular functions, biological processes, and pathways identified may be crucial mechanisms involved in SCI. The top 10 high-degree core proteins were complement component C3 (C3), alpha-2-HS-glycoprotein (Ahsg), T-kininogen 1 (Kng1), Serpinc1 protein (Serpinc1), apolipoprotein A-I (Apoa1), serum albumin (Alb), disulfide-isomerase protein (P4hb), transport protein Sec61 subunit alpha isoform 1 (Sec61a1), serotransferrin (Tf), and 60S ribosomal protein L15 (Rpl15). CONCLUSIONS The proteins identified in this study may provide potential targets for diagnosis and treatment 1 week after SCI.

Figure 1

Heatmap and Volcano plot of the differentially expressed proteins (DEPs). (A) Heatmap of DEPs, in which red represents upregulated DEPs and green represents downregulated DEPs. (B) Volcano plot of DEPs. The X axis is the fold change (log 2), and the Y axis represents the P value (−log 10). Red points (fold change >1.5) indicate upregulated proteins, and blue points (fold change <−1.5) indicate downregulated proteins.

Figure 2

Gene Ontology (GO) term enrichment of differentially expressed proteins (DEPs). (A) All enriched GO terms of the DEPs in the 2 groups of rats. DEPs were categorized according to biological processes, cellular components and molecular functions. (B, C) Enriched terms upregulated (B) and downregulated (C) in the SCI compared with the control group.

Figure 3

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the differentially expressed proteins (DEPs). (A) All enriched KEGG pathways of the DEPs in the 2 groups of rats. (B, C) Enriched pathways of DEPs upregulated (B) and downregulated (C) in the SCI compared with the control group.

Figure 4

Protein–protein interaction (PPI) analysis of differentially expressed proteins (DEPs). Nodes indicate proteins, whereas lines indicate interactions between proteins.

Figure 5

Western blotting of differentially expressed proteins (DEPs). (A) Expression of T-kininogen 1 (Kng1), sodium- and chloride-dependent GABA transporter 3 (Gat3) and sodium/potassium-transporting ATPase subunit beta (Atp1b2) in the SCI and control groups of rats. GAPDH levels were loading control. (B) Quantification of expression of DEPs. * P<0.05.