New insights into cartilage biology and pathology using mouse cartilage proteomics

In recent years there has been an explosion in the use of proteomics to discover novel biomarkers of joint disease, unravel new molecular mechanisms underlying cartilage degeneration and identify cartilage components and interactions that have evaded detection by conventional biochemical approaches. Our focus has been to develop and apply techniques for proteomic analysis of mouse cartilage. Despite the limited amounts of material available, the benefits of mouse tissues outweigh the disadvantages. Specifically, the use of mouse models will facilitate comparison between wild-type tissue with cartilage lacking key elements of the degenerative machinery (e.g. ADAMTS5), cartilage lacking novel or important cartilage components and cartilage harbouring disease (chondrodysplasia)-causing mutations. 2-D electrophoresis (2-DE) remains a popular method for differential proteomics; however the composition of cartilage presents a unique challenge. Using centrifugal ultrafiltration to deplete aggrecan and hyaluronan from the media of femoral head (P21) explant cultures, we identified novel components of the cartilage “secretome”, including proteolytic fragments of perlecan and chondromodulin, and markers for IL-1 and retinoic acid-induced cartilage degeneration, such as lipocalin-2 and fragments of connective tissue growth factor. More recently we developed a fractionation approach based on differential protein solubility for proteomic analysis of cartilage tissue extracts. Sequential extraction of mouse femoral head cartilage with 1 M NaCl followed by 4 M GdnHCl generates very distinct but, importantly, consistent 2-DE profiles. Identification of differentially abundant protein spots using HPLC-MS/MS (Agilent XCTplus ion trap) revealed effective partitioning of readily soluble cytosolic components (e.g. triosephsophate isomerase) from more tightly integrated, predominantly, matrix components (e.g. decorin, matrilin-1, lactadherin). Resolution by 2-DE effectively targets differentially abundant proteins and readily provides a visual map of a tissue proteome (including modified protein isoforms and proteolytic fragments). However, recent advances in high-resolution tandem mass spectrometry have enabled large-scale protein identification from complex peptide mixtures generated by in-solution tryptic digestion. Using nanoLC MS/MS (LTQ-Orbitrap) and spectral counting we identified 600 proteins at high confidence in P21 cartilage extracts, with >250 proteins significantly enriched in either the 1M NaCl or 4M GuHCl extract. Surprisingly, the partitioning of readily and poorly soluble components applied equally to cellular components (e.g. ribonuclear and proteasomal subunits) as ECM proteins and proteoglycans. Our fractionation approach therefore facilitates deeper mining of the cartilage proteome whilst retaining important biochemical information related to the proteins identified. Most recently we have used this approach to identify novel components of cartilage development, using high-density chondrocyte cultures maintained scaffold-free in DMEM/10% FCS/ ascorbic acid for up to 6 weeks. While the mouse “neo-cartilage” lacked stratification, ultrastructural analysis by transmission EM revealed clearly defined zones of pericellular and territorial zones of cartilaginous ECM containing intricate proteoglycan and collagen networks. Comparison of 3-week neo-cartilage with 3-day epiphyseal cartilage by SDS-PAGE revealed major differences in protein extractability. In juvenile cartilage the greater proportion of proteins were readily soluble, whereas in the neo-cartilage a higher proportion of the extract was poorly soluble. Label-free quantitative MS/MS, combined with rigorous statistical and bioinformatic methods, was used to generate “extraction profiles” (NaCl-extracted versus GdnHCl-extracted) of the juvenile cartilage and neo-cartilage proteins. We identified a panel of proteins involved in maturation of the neo-cartilage ECM, i.e. components with the greatest differential in extractability between the two sample types, including many pericellular and extracellular matrix components (e.g. collagen VI, nidogen-2, perlecan, matrilin-3 and COMP). Unexpectedly, one of the guanidine extract specific proteins in the mouse neo-cartilage was a serine protease inhibitor, protease nexin-1. The potential for further applications of cartilage “extraction profiling” in the context of cartilage repair will be discussed.