Development of a multiplexed assay for the targeted analysis of cartilage endogenous peptides in synovial fluid and serum from osteoarthritis patients

Purpose: 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. In the present work, we aimed to develop a targeted method for the quantitative monitoring of this panel of peptides in synovial fluid and serum 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. Samples of human articular cartilage secreted proteins (secretome) 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 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 secretome and synovial fluid to detect those peptides that are better identified in these types of samples, which led to a list of 27 different peptides. Then, a MRM method was designed for the detection of these 27 peptides, belonging to 12 different proteins that were characteristic of human articular cartilage. Table 1 shows the peptides that were detected with high confidence in the secretome of cartilage and also in synovial fluid samples. Conclusions: A multiplexed targeted method for the simultaneous identification of a panel of 17 endogenous peptides from proteins characteristic of articular cartilage has been developed, based on liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometry. By these means, endogenous peptides were detected and relatively quantified in synovial fluid and serum from OA patients. Further qualification studies will be necessary to establish their usefulness for OA diagnosis and progression studies.Table 1Endogenous peptides detected by LC-MRM methodology in the cartilage secretome and synovialProtein nameProtein IDSequence of the cartilage endogenous peptideCartilage oligomeric matrix proteinP49747 (COMP_HUMAN)AEPGIQLKAVSSTGPGEQLRNAVLNQGREIVQTCartilage intermediate layer protein 1O75339 (CILP1_HUMAN)DEGDTFPLRSTATAAQTDLNFINProlarginP51888 (PRELP_HUMAN)DSNKIETIPNSDGVFKPDTFibronectinP02751 (FINC_HUMAN)SSGSGPFTDVRAATSSGSGPFTDVRAADermcidinP81605 (DCD_HUMAN)DAVEDLESVGKENAGEDPGLARMatrix Gla proteinP08493 (MGP_HUMAN)NANTFISPQQRClusterinP10909 (CLUS_HUMAN)TLLSNLEEAKGlia-derived nexinP07093 (GDN_HUMAN)SEDGTKASAATTAI Open table in a new tab