Purpose: In order to understand the pathophysiology of osteoarthritis (OA) its mediators need to be identified. Synovial fluid (SF) is a joint located serum filtrate with additional contributions from articular cartilage, synovium and bone. It represents a potential source of disease specific proteins that could aid in the understanding of the pathogenesis of joint disease and be used in the early diagnosis of disease. Mass spectrometry (MS) provides opportunities to discover disease mechanisms whilst simultaneously identifying potential biomarkers of OA. In MS high abundance proteins interfere with the signal from low abundance proteins; the fraction containing most potential proteins of interest. To overcome this methods of protein depletion or equalisation have been used. However in SF proteomics only protein depletion using expensive immunodepletion columns has been implemented. We therefore investigated the use of a more cost effective equalisation method using ProteominerTM beads in order to comprehensively profile the protein complement of SF in health and OA using liquid chromatography mass spectrometry (LC-MS/MS). Additionally label-free quantification identified potential OA biomarkers. Method(s): Following the collection of SF from the metacarpophalangeal joints of 9 normal and 9 OA racing thoroughbred horses macroscopic, microscopic and synovitis scoring was undertaken. SF was hyaluronidase treated and high abundant proteins depleted using ProteoMinerTM equalisation beads. Reduction, alkylation and trypsin digestion were undertaken directly on the beads after equal protein loading. To investigate the efficiency of ProteoMinerTM technology a single SF sample was analysed without protein depletion. All samples were individually analysed on a two hour gradient with LC-MS/MS using a NanoAcquity LC coupled to a LTQ Orbitrap Velos. ProgenesisTM LC-MS software was used for label-free quantification with data searched for protein identifications using Mascot in the Ensembl database for horse. To maximise the number of quantifiable protein with an acceptable false discovery rate (FDR) the peptide matches were adjusted to 1% FDR prior to the protein identifications being re-imported into ProgenesisTM. Adjusted ANOVA values of p<0.05 and additionally regulation of >2-fold were regarded as significant. Result(s): The average modified Mankins score, palmar osteochondral disease score and synovitis scores for normal samples were 0.8 +/- 0.35, 0 and 1.1 +/-0.2 and OA samples were 13.6 +/- 1.4, 1.7 +/- 0.2, 2.3 +/- 0.2 (mean +/- standard error mean). The number of protein identifications was increased by 33% in the ProteominerTM treated SF compared to undepleted SF. Following ProteominerTMtreatment and ProgenesisTManalysis a total of 754 proteins were identified in SF, 593 with a significant Mascot score. Thus ProteominerTM beads concentrated the lower abundance proteins enabling the most comprehensive SF proteome to date. Proteins identified included those relating to matrix proteins, inflammatory factors, complement activation proteins and proteases. A subset of 10 proteins were identified which were differentially expressed in OA SF (Table 1). Conclusion(s): A number of proteins were identified for the first time in SF which may be involved in the pathogenesis of OA. We identified a distinct set of proteins that may act as potential biomarkers to distinguish between normal and OA joints. S100-A10, a calcium binding protein has upregulated in OA. Other S100 proteins have been demonstrated as having a role in the pathogenesis of OA and in SF proteomic studies. However this is the first time S100-A10 has been implicated in OA. Together with its binding partner annexin 2 it acts as a plasminogen receptor and regulates plasminogen-dependant macrophage activation. This may have a role in the synthesis and activation of matrix degrading proteases. CD109 is a TGF-beta coreceptor, released from the chondrocyte cell surface that inhibits TGF-beta signalling. Its contribution to the disregulation of TGF-beta is unknown. (Table Presented).
CITATION STYLE
Peffers, M., Riggs, C., McDermott, B., & Clegg, P. (2014). Comprehensive Protein Profiling of Synovial Fluid in Osteoarthritis. Equine Veterinary Journal, 46(S47), 19–19. https://doi.org/10.1111/evj.12323_42
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