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. 2024 Mar 27:17:1313-1326.
doi: 10.2147/JPR.S449044. eCollection 2024.

Tandem Mass Tag-Based Proteomic Analysis of Normal and Degenerated Human Intervertebral Discs

Yang Fu #  1 , Xiao-Qin Huang #  1 , Hang-Bo Qu  1 , Yan Zhi Ge  1 , Xuan-Liang Ru  1
Affiliations

Tandem Mass Tag-Based Proteomic Analysis of Normal and Degenerated Human Intervertebral Discs

Yang Fu et al. J Pain Res. .

Abstract

Background: Intervertebral disc degeneration (IVDD) is the main cause of low back pain (LBP), but the specific regulatory factors, pathways and specific molecular mechanisms remain unclear.

Methods: We identified and quantitatively analyzed Pfirrmann Grade II (n=3) and Pfirrmann Grade IV (n=3) pulposus samples via MRI. The differential abundance of proteins in the samples was determined and quantitatively analyzed by relative and absolute quantitative analysis of the isotope marker levels combined with the liquid chromatography-tandem mass spectrometry (LC‒MSMS/MS).

Results: A total of 70 proteins (30 significantly increased proteins (> 1.2-fold change) and 40 significantly decreased proteins (< 0.8-fold change)) showed different levels among the groups. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO) enrichment analyses and Western blot analysis showed that CYCS, RAC1, and PSMD14 may play important roles in IVDD and that Epstein‒Barr virus infection, viral myocarditis, colorectal cancer, nonalcoholic fatty liver disease (NAFLD) and amyotrophic lateral sclerosis (ALS) are the main pathways involved in IVDD.

Conclusion: CYCS, RAC1 and PSMD14 may play important roles in IVDD, and Epstein‒Barr virus infection, viral myocarditis, colorectal cancer, NAFLD and ALS may be the main pathways involved in IVDD.

Keywords: LC‒MS/MS; TMT; low back pain; nucleus pulposus; proteomics.

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Conflict of interest statement

The authors declare that they have no competing interests in this work.

Figures

Figure 1
Figure 1
Heatmap of DEPs. Each row represents a protein, each column represents a sample/repeat, and each color represents a different expression level (log10 for quantification and median correction). The P value was not adjusted for the false discovery rate.
Figure 2
Figure 2
Key target network of the downregulated DEPs in the severe and mild groups. The line represents a protein interaction recorded or predicted by STRING, and each box represents a key protein recorded by Cytoscape.
Figure 3
Figure 3
Key network of upregulated DEPs in the severe and mild groups.
Figure 4
Figure 4
GO annotations for functional classification.
Figure 5
Figure 5
GO enrichment of functional terms. The size of the bubble represents the number of proteins with the GO classification. Fisher’s exact test P value: the enrichment test P value was obtained using Fisher’s exact test; −log10 (P value): logarithmically converted P value obtained with Fisher’s exact test. The P value was not adjusted for the false discovery rate.
Figure 6
Figure 6
KEGG pathway annotations of DEPs. The red bars indicate upregulated pathways, and the green bars indicate downregulated pathways.
Figure 7
Figure 7
KEGG pathway enrichment of DEPs. The bubble size indicates the number of DEPs in the KEGG pathway. Fisher’s exact test P value: enrichment P value obtained with Fisher’s exact test; −log10 (P value): logarithmically converted P value obtained with Fisher’s exact test. The P value was not adjusted for the false discovery rate.
Figure 8
Figure 8
Epstein‒Barr virus infection. The upregulated DEPs CYCS, 19S, and RAC1, which are highlighted in the blue frame, participate in Epstein‒Barr virus infection.
Figure 9
Figure 9
Analysis of the expression of related proteins in different groups. The CYCS, RAC1, PSMD14 and COPS4 levels were analyzed via Western blotting.
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