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Home / Equine Research Bank / J Proteomics / High throughput proteomic analysis of the secretome in an ex...
Journal of proteomics2011; 74(5); 704-715; doi: 10.1016/j.jprot.2011.02.017

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.
Copyright © 2011 Elsevier B.V. All rights reserved.
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Publication Date: 2011-02-24 PubMed ID: 21354348PubMed Central: PMC3078332DOI: 10.1016/j.jprot.2011.02.017Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research project used advanced proteomic techniques to identify the main proteins present in the secretome of articular cartilage under different conditions, and considered implications for understanding early stages of osteoarthritis.

Experimental Design

  • The researchers extracted explants from equine metacarpophalangeal joints. An explant refers to a tissue sample that has been removed from the body of an organism and kept alive in an artificial environment for experimental purposes.
  • The explants were then subjected to different conditions: (i) incubated alone, (ii) with the pro-inflammatory cytokine, interleukin-1beta (IL-1β), or (iii) with IL-1β in the presence of carprofen, a non-steroidal anti-inflammatory drug. Carprofen is employed here to evaluate its effect on the secretome under inflammatory conditions.

Proteomic Analysis

  • The culture medium supernatants (liquid part above the settled cells) were sampled and subjected to a specific protease (trypsin) digestion, breaking down the proteins into smaller peptide fragments.
  • These peptides were then separated by High-Performance Liquid Chromatography (HPLC), a method commonly used to separate components of a mixture, and detected using an ion trap instrument (specifically a Bruker amaZon model). Ion trap is a type of mass spectrometer that uses electric or magnetic fields to trap charged particles.
  • The most abundant peptides in each Mass Spectrometry (MS) scan were fragmented further and their patterns compared with mammalian entries in the Swiss-Prot database, a manually curated protein sequence database. The search engine Mascot was employed for this comparison.

Major Findings

  • Peptides derived from several proteins including aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin and others were found. These are important structural proteins in the cartilage’s extracellular matrix.
  • Quantitative western blotting, a technique that enables detection and measurement of specific proteins, confirmed the presence of several proteins including CILP-1, CLU, MMP-1, MMP-3, and TSP-1.
  • Under inflammatory conditions (interleukin-1beta exposure), the levels of certain proteins (MMP-1, MMP-3, TSP-1) increased, while the precursor for CLU decreased. These proteins are generally involved in cartilage degradation and matrix remodelling processes, which are prominent factors in osteoarthritis development.

Conclusion

  • The findings give significant insight into the proteins active in the inflammatory response of articular cartilage. Understanding these proteins can be instrumental in studying early stages of osteoarthritis, giving potential possibilities for earlier diagnosis and intervention strategies.

Cite This Article

APA
Clutterbuck AL, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A. (2011). High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation. J Proteomics, 74(5), 704-715. https://doi.org/10.1016/j.jprot.2011.02.017

Publication

Journal of proteomics
ISSN: 1876-7737
NlmUniqueID: 101475056
Country: Netherlands
Language: English
Volume: 74
Issue: 5
Pages: 704-715

Researcher Affiliations

Clutterbuck, Abigail L
  • 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.
Smith, Julia R
    Allaway, David
      Harris, Pat
        Liddell, Susan
          Mobasheri, Ali

            MeSH Terms

            • Animals
            • Anti-Inflammatory Agents, Non-Steroidal / pharmacology
            • Carbazoles / pharmacology
            • Cartilage, Articular / metabolism
            • Cartilage, Articular / pathology
            • Gene Expression Regulation
            • Horses
            • Inflammation / drug therapy
            • Inflammation / metabolism
            • Inflammation / pathology
            • Models, Biological
            • Osteochondritis / drug therapy
            • Osteochondritis / metabolism
            • Osteochondritis / pathology
            • Proteome / metabolism

            Grant Funding

            • BBS/S/M/2006/13141 / Biotechnology and Biological Sciences Research Council

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