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

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-steroid...

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Autores principales: Clutterbuck, Abigail L., Smith, Julia R., Allaway, David, Harris, Pat, Liddell, Susan, Mobasheri, Ali
Formato: Texto
Lenguaje:English
Publicado: Elsevier 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078332/
https://www.ncbi.nlm.nih.gov/pubmed/21354348
http://dx.doi.org/10.1016/j.jprot.2011.02.017
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author Clutterbuck, Abigail L.
Smith, Julia R.
Allaway, David
Harris, Pat
Liddell, Susan
Mobasheri, Ali
author_facet Clutterbuck, Abigail L.
Smith, Julia R.
Allaway, David
Harris, Pat
Liddell, Susan
Mobasheri, Ali
author_sort Clutterbuck, Abigail L.
collection PubMed
description 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.
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spelling pubmed-30783322011-06-28 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 J Proteomics Article 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. Elsevier 2011-05-01 /pmc/articles/PMC3078332/ /pubmed/21354348 http://dx.doi.org/10.1016/j.jprot.2011.02.017 Text en © 2011 Elsevier B.V. https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Article
Clutterbuck, Abigail L.
Smith, Julia R.
Allaway, David
Harris, Pat
Liddell, Susan
Mobasheri, Ali
High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
title High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
title_full High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
title_fullStr High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
title_full_unstemmed High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
title_short High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
title_sort high throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078332/
https://www.ncbi.nlm.nih.gov/pubmed/21354348
http://dx.doi.org/10.1016/j.jprot.2011.02.017
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