Preparation and Application of a Decellularized Extracellular Matrix for Identification of ADAMTS Substrates
Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.
Key wordsDecellularized extracellular matrix Substrate identification ADAMTS protease Mass spectrometry
This work was supported by a grant from The Swiss National Science Foundation (SNF, Project funding in biology and medicine (division III)) grant no. 31003A_156740.
- 10.Bekhouche M, Leduc C, Dupont L, Janssen L, Delolme F, Vadon-Le Goff S, Smargiasso N, Baiwir D, Mazzucchelli G, Zanella-Cleon I, Dubail J, De Pauw E, Nusgens B, Hulmes DJ, Moali C, Colige A (2016) Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-beta signaling as primary targets. FASEB J 30:1741–1756CrossRefGoogle Scholar
- 11.Schnellmann R, Sack R, Hess D, Annis DS, Mosher DF, Apte SS, Chiquet-Ehrismann R (2018) A selective extracellular matrix proteomics approach identifies fibronectin proteolysis by a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS16) and its impact on spheroid morphogenesis. Mol Cell Proteomics 17:1410–1425CrossRefGoogle Scholar
- 14.Gao G, Plaas A, Thompson VP, Jin S, Zuo F, Sandy JD (2004) ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1. J Biol Chem 279:10042–10051CrossRefGoogle Scholar
- 15.Beacham DA, Amatangelo MD, Cukierman E (2007) Preparation of extracellular matrices produced by cultured and primary fibroblasts. Curr Protoc Cell Biol Chapter 10, Unit 10.19Google Scholar