Background The low-molecular-weight subset of the cartilage proteome (also termed cartilage peptidome) may have a diagnostic potential in osteoarthritis (OA) research, not yet fully exploited.
Objectives The aim of this study is to investigate the cartilage secretome by means of peptidomic analysis and to provide a novel source for OA biomarker discovery.
Methods Tissue explants were obtained from the dissection of human OA cartilages, both from the unwounded zone (UZ) and the wounded zone (WZ). The study was approved by the local ethical committee. Cartilage shavings from each zone were cut into 6 mm discs and five discs/zone were placed into 96 wells plates and incubated (37 °C/5% CO2) in serum-free DMEM up to 6 days. Conditioned media from each condition were collected at day 1, 3 and 6. Selective extraction of endogenous peptides was accomplished by combining ultrafiltration and solid phase extraction. Then, the peptides were directly analyzed by nano reversed-phase liquid chromatography (RP-LC) in an Easy nLC II system coupled to an ion trap LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific). Peptide/protein identifications were searched against database (human_fasta) using the SEQUEST algorithm (Proteome Discoverer 1.3, Thermo Scientific).
Results After optimization steps of the procedure, day 3 was selected as the best point for our proteomic analysis to compare the peptide profiles from UZ and WZ, showing the highest number of unique peptides/proteins and the lowest serum contamination. Globally, this study led to the identification of a panel of 262 peptides corresponding to 36 proteins that were differentially released from the UZ and WZ in OA cartilage. 60 peptides (corresponding to 20 proteins) were detected only in the UZ (early biomarkers), while 18 peptides (corresponding to 5 proteins) only in the WZ (late biomarkers). Finally, 11 proteins were detected in both zones; however also in this case the number of peptides was higher in the UZ than in the WZ. These results suggest that cartilage from the UZ shows a protein turnover rate higher than cartilage from the WZ, thus facilitating the discovery of OA biomarkers in an early stage of the disease, when radiographic signs are not yet detectable. Data mining revealed that most of the identified proteins were cartilage ECM proteins or proteins with well-established matrix functions, such as collagens and proteoglycans, thus indicating the high cartilage-specific biomarker concentration present in our samples. Some of these proteins have been associated with OA pathology before (collagen II, proteoglycan 4, tubulin, vimentin), while others are described here for the first time (augurin, salivary acidic proline-rich phosphoprotein 1/2, dermcidin).
Conclusions Our results clearly indicate that profiling of peptides from cartilage secretome represents a promising alternative in the field of OA biomarker discovery. In this study we highlight its potential application as novel OA biomarker source both for early disease detection (early biomarkers) and for monitoring disease progression (late biomarkers). Further investigations of these peptides on biological fluids will assess their biomarker usefulness.
Disclosure of Interest None declared
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