Proteomic profiling of cartilage ECM formation and maturation using sequential protein extraction and quantitative mass spectrometry
Wilson, RR and Diseberg, A and Gorman, J and Bateman, J, Proteomic profiling of cartilage ECM formation and maturation using sequential protein extraction and quantitative mass spectrometry, FECTS, July 3-7, 2010, Davos, Switzerland (2010) [Conference Edited]
Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neo-cartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neo-cartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neo-cartilage has not been investigated by proteomics. To redress this, we developed a mouse neo-cartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neo-cartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neo-cartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility, from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (GuHCl-extracted) proteins in neo-cartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis was then used to filter three significant protein groups: proteins enriched according to extraction condition; proteins differentially abundant between juvenile cartilage and neo-cartilage, and proteins with differential solubility properties between the two tissue types. Bioinformatic classification of proteins differentially abundant between NaCl and GuHCl extracts (n = 403) revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neo-cartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (EDIL3, CCD80, EMIL1 and PEDF). Proteins with differential extractability between juvenile cartilage and neo-cartilage included ECM components (NID2, HSPG2, CO6A1, MATN3, TENA and TSP1) and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neo-cartilage protein, protease nexin-1 (PN-1), was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates PN-1 in cartilage development in vitro and in vivo.