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Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, Cheshire, CH64 7TE, UK
Protein Function Group, Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7ZB, UK
These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 21 June 2013; in revised form: 19 August 2013 / Accepted: 23 September 2013 / Published: 15 October 2013
Abstract: Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA.
Keywords: cartilage; human; osteoarthritis; QconCAT; secretome
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Peffers, M.J.; Beynon, R.J.; Clegg, P.D. Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome. Int. J. Mol. Sci. 2013, 14, 20658-20681.
Peffers MJ, Beynon RJ, Clegg PD. Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome. International Journal of Molecular Sciences. 2013; 14(10):20658-20681.
Peffers, Mandy J.; Beynon, Robert J.; Clegg, Peter D. 2013. "Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome." Int. J. Mol. Sci. 14, no. 10: 20658-20681.