High-throughput proteomic analysis of the cartilage secretome for identification of inflammatory biomarkers

Abstract

digestion by trypsin and reversed phase separations coupled to tandem mass spectrometry for the identification and targeted quantification of peptides by multiple reaction monitoring (MRM). The tissue was sliced from synovial surface to bone in 10mm thin sections and the full thickness cartilage was divided into 100mm pools including superficial, intermediate and deep zones. Results: Previous known distribution of proteoglycan 4 (PRG4, lubricin, superficial zone protein) was confirmed but also novel findings were observed e.g. asporin which was predominantly present in the superficial layers. In total 40 proteins were quantified showing distinct patterns in normal tissue. In addition we observed altered protein distribution patterns in preclinical (macroscopically normal) tissue from a joint with early fibrillation (OA-lesions) on the opposite surface. Conclusions: As an alternative to immunohistochemistry we used proteomics technology to study the protein abundance across full thickness articular cartilage. The advantages of this approach are that it allows multiple targets to be studied simultaneously, it is independent of antibody availability and circumvent some antibody-related artifacts. Other advantages include unambiguous identifications and improved quantifications. The work shows novel information on the differences that exist in different layers of cartilage that is of value in understanding changes in early pathology.