Discussion

Peffers, M.J., Beynon, R.J., Thornton D.J., Clegg, P.D
Aims: Articular cartilage is composed of a single cell type, the chondrocyte, embedded within an extracellular matrix (ECM) the composition and structure of which provides its properties. Osteoarthritis (OA) is characterised by the slow degeneration of cartilage ECM. We used comprehensive proteomic analysis methodologies on the cartilage ECM to identify low abundance proteins. Then, QconCAT technology allowed parallel quantification of large sets of analyte proteins, to quantify for the first time the major matrix proteins using mass-spectrometry. Methods: Full thickness equine articular cartilage (n = 10) was harvested and sequential extractions using NaCl and guanidine hydrochloride were undertaken. Mass spectrometric analysis was commenced following reduction, alkylation and trypsin digestion by LC-MS/MS using a NanoAcquity LC coupled to a LTQ Velos. Data produced were searched using Mascot (Matrix Science UK), against the Equus caballus database. An equine cartilage QconCAT was designed as a concatenation of tryptic quantotypic peptides using peptides identified here. Analyte and QconCAT were reduced, alkylated and trypsin-digested in solution on 10,000 MWCO centrifugal, peptide mixtures were resolved by LC- MS using a Waters Xevo-triple quadropole-mass spectrometer. Ratios were normalised to dry weight of cartilage. Results: Five hundred and eighty-five proteins were identified using sequential extraction techniques including both matrix and chondrocytes proteins. Selected matrix proteins were then quantified using the QconCAT. Matrix proteins were quantified as pmol/mg dry weight of cartilage: aggrecan 108.68 plusminus 16.2 s.e.m., biglycan 693.7 plusminus 122 s.e.m., decorin 1173.6 plusminus 1166 s.e.m. and COMP 77.2 plusminus 11.6 s.e.m. Conclusions: This study enabled the characterisation and absolute quantitation of equine cartilage ECM using QconCAT technology. Quantification data for the proteins studied will enable baseline parameters to be set for cartilage matrix components enabling the study of arthritic pathology and physiological ageing and may help in biomarker application. Acknowledgements: Mandy Peffers is funded by a Wellcome Integrated Research Training Fellowship.