title: High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation.
abstract: This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1β increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis.
authors.affiliation: Musculoskeletal Research Group, Division of Veterinary Medicine, School of Veterinary Medicine and Science, Faculty of Medicine and Health Sciences, University of Nottingham, Leicestershire, United Kingdom.
authors.forename: Abigail L
authors.initials: AL
authors.lastname: Clutterbuck
authors.forename: Julia R
authors.initials: JR
authors.lastname: Smith
authors.forename: David
authors.initials: D
authors.lastname: Allaway
authors.forename: Pat
authors.initials: P
authors.lastname: Harris
authors.forename: Susan
authors.initials: S
authors.lastname: Liddell
authors.forename: Ali
authors.initials: A
authors.lastname: Mobasheri
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006736:Horses; D007249:Inflammation / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008954:Models, Biological; D010007:Osteochondritis / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D020543:Proteome / Q000378:metabolism*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D002227:Carbazoles; D020543:Proteome; C007005:carprofen