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

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

Authors

Abigail L. Clutterbuck

Julia R. Smith

David Allaway

Pat Harris

Susan Liddell

Ali Mobasheri



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.

Journal Article Type Article
Publication Date May 1, 2011
Journal Journal of Proteomics
Print ISSN 1874-3919
Electronic ISSN 1874-3919
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 74
Issue 5
APA6 Citation Clutterbuck, A. L., Smith, J. R., Allaway, D., Harris, P., Liddell, S., & Mobasheri, A. (2011). High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation. Journal of Proteomics, 74(5), doi:10.1016/j.jprot.2011.02.017
DOI https://doi.org/10.1016/j.jprot.2011.02.017
Publisher URL http://www.sciencedirect.com/science/article/pii/S1874391911000558
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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