title: Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization.
abstract: OBJECTIVE
  The 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.
  
  
  METHODS
  SF 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Comparative Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, Wirral, CH64 7TE, UK. Electronic address: peffs@liv.ac.uk.
authors.forename: M J
authors.initials: MJ
authors.lastname: Peffers
authors.affiliation: Comparative Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, Wirral, CH64 7TE, UK. Electronic address: benmcd@liverpool.ac.uk.
authors.forename: B
authors.initials: B
authors.lastname: McDermott
authors.affiliation: Comparative Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, Wirral, CH64 7TE, UK. Electronic address: pclegg@liverpool.ac.uk.
authors.forename: P D
authors.initials: PD
authors.lastname: Clegg
authors.affiliation: Hong Kong Jockey Club, Equine Hospital, Sha Tin Racecourse, New Territories, Hong Kong. Electronic address: christopher.m.riggs@hjkc.org.hk.
authors.forename: C M
authors.initials: CM
authors.lastname: Riggs
mesh_terms: D000818:Animals; D017306:Annexin A2 / Q000096:biosynthesis; D002358:Cartilage, Articular / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006734:Horse Diseases / Q000378:metabolism* / Q000473:pathology; D006736:Horses; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology / Q000662:veterinary*; D010446:Peptide Fragments / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D009418:S100 Proteins / Q000096:biosynthesis; D013582:Synovial Fluid / Q000378:metabolism*; D013583:Synovial Membrane / Q000378:metabolism; D000099248:S100 Calcium Binding Protein A10
chemical_list: D017306:Annexin A2; D010446:Peptide Fragments; D020543:Proteome; D012333:RNA, Messenger; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10
keywords: Equalization; Neopeptide; Osteoarthritis; S100-A10; Synovial fluid
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