Background: Are there differences in protein patterns relating to different cartilage properties? Results: Quantitative proteomics of cartilage from articulating joints, trachea, rib and intervertebral disc revealed distinct differences. Conclusion: Observed differences are pronounced between different types of cartilage, whereas less marked significant between subtypes of articular cartilages. Significance: The data provides novel insights into tissue structure-function and tropism of disease.

Galectin-1, a β-galactoside binding lectin involved in immunoregulation and cancer, binds natural and many synthetic multivalent glycoconjugates with an apparent glycoside cluster effect, that is, affinity above and beyond what would be expected from the concentration of the determinant sugar. Here we have analyzed the mechanism of such cluster effects in solution at physiological concentration using a fluorescence anisotropy assay with a novel fluorescent high-affinity galectin-1 binding probe. The interaction of native dimeric and monomeric mutants of rat and human galectin-1 with mono- and divalent small molecules, fetuin, asialofetuin, and human serum glycoproteins was analyzed. Surprisingly, high-affinity binding did not depend much on the dimeric state of galectin-1 and thus is due mainly to monomeric interactions of a single carbohydrate recognition domain. The mechanism for this is unknown, but one possibility includes additional interactions that high-affinity ligands make with an extended binding site on the carbohydrate recognition domain. It follows that such weak additional interactions must be important for the biological function of galectin-1 and also for the design of galectin-1 inhibitors.

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic) and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of OA, OA progression, and the joint specific biomarkers.

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.