Defining Elastic Fiber Interactions by Molecular Fishing AFFINITY PURIFICATION AND MASS SPECTROMETRY APPROACH* □ S

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix. Molecular & Cellular Proteomics 8:2715–2732, 2009. Mass spectrometry is emerging as a powerful approach to identify protein interaction partners in molecular complexes. We have developed an affinity purification and mass spectrometry strategy that is applicable to the analysis of molecular interactions of extracellular matrix complexes. The extracellular matrix provides structural support to tissues and profoundly influences cell survival, proliferation, migration, and phenotypic state. It is a complex multimolecular and three-dimensional milieu that comprises assembled networks of tissue-specific combinations of structural and cell-adhesive glycoproteins, proteoglycans, and cross-linking enzymes. The matrix also sequesters numerous growth factors and cytokines, thereby controlling their bioavailability. Delineating the molecular nature of the fundamental in