Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization

M.J Peffers,B Mcdermott,P.D Clegg,C.M Riggs

doi:10.1016/j.joca.2015.03.019

M.J Peffers, B Mcdermott + Show 2 more

Open Access

https://doi.org/10.1016/j.joca.2015.03.019

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Abstract

SummaryObjectiveThe aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins.DesignSF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. In addition immunohistochemistry, western blotting and mRNA expression analysis was undertaken on selected joint tissues.ResultsThe number of protein identifications was increased by 33% in the ProteoMiner™ treated SF compared to undepleted SF. A total of 764 proteins (462 with≥2 significant peptides) were identified in SF. A subset of 10 proteins were identified which were differentially expressed in OA SF. S100-A10, a calcium binding protein was upregulated in OA and validated with western blotting and immunohistochemistry. Several new OA specific peptide fragments (neopeptides) were identified.ConclusionThe protein equalisation method compressed the dynamic range of the synovial proteins identifying the most comprehensive SF proteome to date. 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 and neopeptides that may act as potential biomarkers to distinguish between normal and OA joints.

Highlights

Osteoarthritis (OA) is a joint disease characterised by alterations in the chondrocytes and loss of cartilage extracellular matrix (ECM)[1], leading to biomechanical failure of articular cartilage
We developed a peptide-based affinity method (ProteoMinerTM) which has been demonstrated to have a high level of reproducibility and consistency in terms of the species and quantities of proteins captured[31]
We demonstrated our novel workflow technique involving peptide-based equalisation followed by liquid chromatography coupled to a high mass resolution mass-spectrometer was appropriate for Synovial fluid (SF) proteomics for both discovery and quantification

Summary

Introduction

Osteoarthritis (OA) is a joint disease characterised by alterations in the chondrocytes and loss of cartilage extracellular matrix (ECM)[1], leading to biomechanical failure of articular cartilage. Delayed diagnosis precludes preventive or timely therapeutic interventions. These limitations could be surmounted by enhanced knowledge of articular tissue metabolism and homoeostasis. Synovial fluid (SF) is located in joint cavities and comprises a serum filtrate with additional contributions from surrounding tissues. As it is in direct contact with the OA-affected tissues it has been implicated as a contributor to OA pathophysiology. It represents a potential source of disease-specific proteins that could both aid in the understanding of the pathogenesis of joint disease and act as disease biomarkers.

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