High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation

  • Clutterbuck A
  • Smith J
  • Allaway D
 et al. 
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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, 10. ng/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. © 2011 Elsevier B.V.

Author-supplied keywords

  • Articular cartilage
  • Explant culture
  • High-throughput proteomics
  • Mass spectrometry
  • Osteoarthritis (OA)
  • Secretome

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Authors

  • Ali MobasheriState Research Institute Centre for Innovative Medicine

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  • Abigail L. Clutterbuck

  • Julia R. Smith

  • David Allaway

  • Pat Harris

  • Susan Liddell

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