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

Abstract

ObjectivesTo compare the proteomics of synovial fluid (SF)-derived exosomes in rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), gout, and osteoarthritis (OA) patients.MethodsExosomes 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.ResultsA 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).ConclusionOur 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).

The biomarkers discovery of hyperuricemia and gout: proteomics and metabolomics

“…The presence of significantly expressed inflammatory proteins in gout patients suggests that the inflammatory mechanism deserves our attention ( Shen et al, 2021a ; Qiu et al, 2021 ). As a final observation, patients with gout showed an even more profound association with complement and coagulation cascade pathways ( Shen et al, 2021a ; Chen et al, 2021a ; Huang et al, 2022 ). In summary, multiple biomarkers can provide insights into disease mechanisms based on their expression and functions through a holistic approach to identifying diseases.…”

“…There are 25 highly unique expressed proteins, including lysozyme C, alpha-1-acid glycoprotein 1, lactotransferrin, S100A9, and myeloperoxidase. They may play a role in neutrophil degranulation, prion diseases, as well as complement and coagulation cascade complement cascades ( Huang et al, 2022 ). Similarly, Qiu et al (2021) found that differentially expressed genes in the male gout group were predominantly enriched in the inflammatory response, S100 protein binding.…”

How to process synovial fluid samples of gouty arthritis and extract its exosomes for subsequent cytokine analysis

“…It has been demonstrated in a recent study that the separation and purification of synovial fluid-derived exosomes can be achieved using the ExoQuick chemical reagent method, whereby 69 differentially expressed proteins such as lysozyme C and protein S100-A9 in GASF-derived exosomes were isolated, and compared with osteoarthritis, axial ankylosing spondylitis, and rheumatoid arthritis synovial fluid-derived exosomes. [9]…”

“…To date, no studies comparing the characteristics and inflammatory effects of GASF-derived exosome among different extraction methods have been published. [9]…”

“…[7,8] SF-derived exosomes enrichment is currently accomplished by various methods, including ultracentrifugation (UC) and polymer-based precipitation methods, among others. Recently, scholars [9,10] have investigated gouty arthritis synovial fluid (GASF)-derived exosomes using the chemical precipitation method (e.g. commercial kits), but few have been concerned with the different extraction methods which determine the purification rate of GASF-derived exosomes.…”