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Proteomic profiling of cartilage ECM formation and maturation using sequential protein extraction and quantitative mass spectrometry

conference contribution
posted on 2023-05-24, 11:27 authored by Richard WilsonRichard Wilson, Diseberg, A, Gorman, J, Bateman, J
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.


Publication title

17th Meeting of the Federation of European Connective Tissues Societies



Event title

17th Meeting of the Federation of European Connective Tissues Societies

Event Venue

Davos, Switzerland

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Date of Event (End Date)


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Expanding knowledge in the health sciences

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