Background Peptidomics is an emerging field branching from proteomics that targets endogenously produced protein fragments. A peptidomic analysis that we recently performed enabled the identification of endogenous peptides that were differentially released from wounded (WZ) and unwounded (UZ) zones of human articular osteoarthritic cartilage, compared to healthy tissue.
Objectives In the present work, we aimed to develop a targeted method for the quantitative monitoring of this panel of peptides in synovial fluids and sera from osteoarthritis (OA) patients, in order to unravel the putative biomarker value of these molecules.
Methods Synovial fluid and serum samples were obtained from OA patients at different stages of the disease. Proteins secreted from human articular cartilage (secretomes) were obtained by culture of tissue explants, according to optimized procedures . The enrichment of endogenous peptides in these three types of samples was standardized, using ultrafiltration procedures and solid phase extraction (SPE) with reversed phase (C18) resins. A method for the targeted identification of endogenous peptides by Multiple Reaction Monitoring (MRM)-mass spectrometry was developed employing cartilage secretome samples, and then applied on synovial fluid and serum. The peptides were separated by nano-LC coupled to a 5500 QTRAP mass spectrometer. Data analysis was performed using the Skyline software. Peptide identifications were searched against a human database (human_fasta) using the ProteinPilot program.
Results The peptidomic study led to the identification of a panel of 262 peptides corresponding to 36 different proteins that were differentially released from the WZ and UZ in OA cartilage. From these, we first performed independent data analyses (IDA) by mass spectrometry using cartilage secretomes and synovial fluids to detect those peptides that are better identified in these types of samples, which led to a list of 27 different peptides. Then, a final MRM method was designed for the detection of these peptides, belonging to unique proteins that were characteristic of human articular cartilage.
Conclusions A multiplexed targeted method for the simultaneous identification of a panel of 17 endogenous peptide released from 7 proteins characteristic of articular cartilage has been developed. The proteins detected are Cartilage oligomeric matrix, Cartilage intermediate layer protein 1, Fibronectin, Prolargin, Dermcidin, Matrix gla protein, Clusterin and Glia-derived nexin. This method is based on liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometry. By these means, endogenous peptides were simultaneously detected and relatively quantified in synovial fluids and sera from OA patients and controls. Further qualification studies will be necessary to establish their usefulness for OA diagnosis and progression studies.
Lourido L, Calamia V, Mateos J, Fernández-Puente P, Fernández-Tajes J, Blanco FJ, Ruiz-Romero C. Quantitative proteomic profiling of human articular cartilage degradation in osteoarthritis. J Proteome Res. 2014; 13(12):6096–106.
Disclosure of Interest None declared