Proteomic analysis of human synovial fluid reveals potential diagnostic biomarkers for ankylosing spondylitis

Abstract Background Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting the axial skeleton and peripheral joints. The etiology of this disease remains poorly understood, but interactions between genetic and environmental factors have been implicated. The present study identified differentially expressed proteins in the synovial fluid (SF) of AS patients to elucidate the underlying cause of AS. Methods A cohort of 40 SF samples from 10 AS and 10 each of rheumatoid arthritis (RA), gout, and osteoarthritis (OA) patients were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins specific to AS. The label-free LC-MS/MS results were verified by western blotting. Results We identified 8 proteins that were >1.5-fold upregulated in the SF of AS patients compared to that of the disease control groups, including HP, MMP1, MMP3, serum amyloid P-component (APCS), complement factor H-related protein 5 (CFHR5), mannose-binding lectin 2 (MBL2), complement component C9 (C9), and complement C4-A (C4A). CFHR5 and C9 were previously found in serum from AS patients, while APCS was previously found in SF as well as in serum. However, the present study has identified C4A, and MBL2 as potential AS biomarkers for the first time. The expression levels of MMP3, C9, and CFHR5 were verified in AS SF using western blotting. Conclusion We performed quantitative comparative proteomic analysis using by LC-MS/MS of the SF from four disease states: RA, gout, and OA. This systematic comparison revealed novel differentially expressed proteins in AS SF, as well as two previously reported candidate biomarkers. We further verified the expression of MMP3, C9 and CFHR5 by western blot. These proteins may serve as diagnostic or prognostic biomarkers in patients with AS, and may thus improve the clinical outcomes of this serious disease.

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Proteomic analysis of human synovial fluid reveals potential diagnostic biomarkers for ankylosing spondylitis

10.21203/rs.2.19736/v1 ◽

2020 ◽

Author(s):

Ji Hyun Lee ◽

Jae Hun Jung ◽

Jeesoo Kim ◽

Won-Ki Baek ◽

Jinseol Rhee ◽

...

Keyword(s):

Ankylosing Spondylitis ◽

Synovial Fluid ◽

Western Blotting ◽

Proteomic Analysis ◽

Axial Skeleton ◽

Factor H ◽

Differentially Expressed ◽

Complement Factor ◽

Differentially Expressed Proteins ◽

Label Free

Abstract Background Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting the axial skeleton and peripheral joints. The etiology of this disease remains poorly understood, but interactions between genetic and environmental factors have been implicated. The present study identified differentially expressed proteins in the synovial fluid (SF) of AS patients to elucidate the underlying cause of AS. Methods A cohort of 40 SF samples from 10 AS and 10 each of rheumatoid arthritis (RA), gout, and osteoarthritis (OA) patients were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins specific to AS. The label-free LC-MS/MS results were verified by western blotting. Results We identified 8 proteins that were >1.5-fold upregulated in the SF of AS patients compared to that of the disease control groups, including HP, MMP1, MMP3, serum amyloid P-component (APCS), complement factor H-related protein 5 (CFHR5), mannose-binding lectin 2 (MBL2), complement component C9 (C9), and complement C4-A (C4A). CFHR5 and C9 were previously found in serum from AS patients, while APCS was previously found in SF as well as in serum. However, the present study has identified C4A, and MBL2 as potential AS biomarkers for the first time. The expression levels of MMP3, C9, and CFHR5 were verified in AS SF using western blotting. Conclusion We performed quantitative comparative proteomic analysis using by LC-MS/MS of the SF from four disease states: RA, gout, and OA. This systematic comparison revealed novel differentially expressed proteins in AS SF, as well as two previously reported candidate biomarkers. We further verified the expression of MMP3, C9 and CFHR5 by western blot. These proteins may serve as diagnostic or prognostic biomarkers in patients with AS, and may thus improve the clinical outcomes of this serious disease.

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The influence of sample collection, handling and low temperature storage upon NMR metabolic profiling analysis in human synovial fluid

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2021.113942 ◽

2021 ◽

Vol 197 ◽

pp. 113942

Author(s):

Matthew K.J. Jaggard ◽

Claire L. Boulangé ◽

Gonçalo Graça ◽

Pouya Akhbari ◽

Uddhav Vaghela ◽

...

Keyword(s):

Synovial Fluid ◽

Low Temperature ◽

Metabolic Profiling ◽

Sample Collection ◽

Low Temperature Storage ◽

Metabolic Profiling Analysis ◽

Profiling Analysis ◽

Human Synovial Fluid ◽

Temperature Storage

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Isolation of human synovial-fluid hyaluronate by density-gradient ultracentrifugation and evaluation of its protein content

Biochemical Journal ◽

10.1042/bj1261073 ◽

1972 ◽

Vol 126 (5) ◽

pp. 1073-1080 ◽

Cited By ~ 30

Author(s):

Irwin Scher ◽

David Hamerman

Keyword(s):

Synovial Fluid ◽

Density Gradient ◽

Density Gradient Ultracentrifugation ◽

Proteolytic Digestion ◽

Peptide Fragments ◽

Guanidinium Chloride ◽

Caesium Chloride ◽

Normal Human ◽

Protein Moiety ◽

Human Synovial Fluid

1. A compound of hyaluronate and protein, called hyaluronate–protein was isolated from pooled human synovial fluids by caesium chloride density-gradient ultracentrifugation. 2. The isolated hyaluronate–protein was labelled with [125I]iodide and the following studies were done. (a) Ultracentrifugation in caesium chloride showed that the protein moiety (125I counts) and hyaluronate (hexuronate) sedimented together in the middle of the gradient. (b) The labelled hyaluronate–protein was treated with trypsin, and ultracentrifugation showed that peptide fragments (125I counts) were dispersed throughout the gradient, indicating proteolytic digestion. Hyaluronate sedimented in the middle of the gradient. (c) The labelled hyaluronate–protein was digested with streptococcal hyaluronidase, and ultracentrifugation showed that hyaluronate fragments were dispersed throughout the gradient, indicating digestion of the polysaccharide. The protein moiety, without attached hyaluronate, now sedimented at the top of the gradient. (d) Ultracentrifugation of labelled hyaluronate–protein in 4m-guanidinium chloride showed that protein and hyaluronate sedimented together. 3. These studies confirm that hyaluronate is combined with a small quantity of protein in normal human synovial fluid. A mild method for the rapid isolation of hyaluronate–protein in good yield is described.

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