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. 2022 Mar 11:13:800902.
doi: 10.3389/fimmu.2022.800902. eCollection 2022.

TMT-Based Quantitative Proteomics Analysis of Synovial Fluid-Derived Exosomes in Inflammatory Arthritis

Yukai Huang  1 , Yuqi Liu  1   2 , Qidang Huang  1 , Shanmiao Sun  1 , Zhuyi Ji  1 , Lixin Huang  1 , Zhi Li  1 , Xuechan Huang  1 , Weiming Deng  1 , Tianwang Li  1   2   3
Affiliations

TMT-Based Quantitative Proteomics Analysis of Synovial Fluid-Derived Exosomes in Inflammatory Arthritis

Yukai Huang et al. Front Immunol. .

Abstract

Objectives: To compare the proteomics of synovial fluid (SF)-derived exosomes in rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), gout, and osteoarthritis (OA) patients.

Methods: Exosomes were separated from SF by the Exoquick kit combined ultracentrifugation method. Tandem mass tags (TMT)-labeled liquid chromatography mass spectrometry (LC-MS/MS) technology was used to analyze the proteomics of SF-derived exosomes. Volcano plot, hierarchical cluster, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted.

Results: A total of 1,678 credible proteins were detected. Sixty-nine differentially expressed proteins were found in gout, compared with OA, axSpA, and RA simultaneously. Twenty-five proteins were found highly expressed in gout uniquely, lysozyme C and protein S100-A9 included, whose bioinformatic analysis was significantly involved in "neutrophil degranulation" and "prion diseases". Eighty-four differentially expressed proteins were found in axSpA, compared with OA, gout, and RA simultaneously. Thirty-nine proteins were found highly expressed in axSpA uniquely, RNA-binding protein 8A and protein transport protein Sec24C included, whose bioinformatic analysis was significantly involved in "acute-phase response" and "citrate cycle". One hundred and eighty-four differentially expressed proteins were found in RA, compared with OA, gout, and axSpA simultaneously. Twenty-eight proteins were found highly expressed in RA uniquely, pregnancy zone protein (PZP) and stromelysin-1 included, whose bioinformatic analysis was significantly involved in "serine-type endopeptidase inhibitor activity" and "complement and coagulation cascades". Enzyme-linked immunosorbent assay (ELISA) result showed that the exosome-derived PZP level of SF in RA was higher than that in OA (p < 0.05).

Conclusion: Our study for the first time described the protein profiles of SF-derived exosomes in RA, axSpA, gout, and OA patients. Some potential biomarkers and hypothetical molecular mechanisms were proposed, which may provide helpful diagnostic and therapeutic insights for inflammatory arthritis (IA).

Keywords: biomarkers; exosomes; inflammatory arthritis; proteomics analysis; synovial fluid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The isolation and identification of SF-derived exosomes. (A–D) The size of exosomes detected by HSFC for nanoparticle analysis (A: Gout; B: axSpA; C: OA; D: RA). (E) The comparison of exosome concentrations in the four groups based on HSFC for nanoparticle analysis. (F) The expression of CD81 and TSG101 was detected by Western blot. *p-value < 0.05 vs. gout group, # p-value < 0.05 vs. other groups.
Figure 2
Figure 2
Quality control of proteomics data. (A) PCA analysis. (B) Box plot analysis. (C) Density map analysis. (D) Corrplot analysis. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Overall distribution of differentially expressed proteins. (A–F) The differentially expressed proteins analyzed by volcano plots between every two groups (A: OA vs. Gout; B: axSpA vs. OA; C: RA vs. axSpA; D: axSpA vs. Gout; E: RA vs. Gout; F: RA vs. OA). (G) Hierarchical clustering analysis of the differentially expressed proteins in the four groups. In the color bar, red represents high expression, and purple represents low expression.
Figure 4
Figure 4
The screening and function analysis of proteins highly expressed in gout. (A) Venn diagram analysis of OA vs. Gout, axSpA vs. Gout, and RA vs. Gout. (B) Hierarchical clustering analysis of the differentially expressed proteins. (C) GO analysis of the highly expressed proteins. (D) KEGG pathway analysis of the highly expressed proteins.
Figure 5
Figure 5
The screening and function analysis of proteins highly expressed in axSpA. (A) Venn diagram analysis of axSpA vs. OA, axSpA vs. Gout, and RA vs. axSpA. (B) Hierarchical clustering analysis of the differentially expressed proteins. (C) GO analysis of the highly expressed proteins. (D) KEGG pathway analysis of the highly expressed proteins.
Figure 6
Figure 6
The screening and function analysis of proteins highly expressed in RA. (A) Venn diagram analysis of RA vs. OA, RA vs. Gout, and RA vs. axSpA. (B) Hierarchical clustering analysis of the differentially expressed proteins. (C) GO analysis the highly expressed proteins. (D) KEGG pathway analysis of the highly expressed proteins.
Figure 7
Figure 7
Verification of exosome-derived PZP in SF by ELISA. The ELISA kits were performed to detect the PZP level of SF-derived exosomes in gout, axSpA, OA, and RA. *P value < 0.05 vs. OA groups.
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