Myofibroblasts form adhesions to their underlying extracellular matrices, which is an essential step in their formation and differentiation. These adhesions comprise protein-rich aggregates of a wide variety of signaling, cytoskeletal, cell adhesion, and matrix proteins that interact with one another to enable bidirectional flow of information between the cell and the surrounding extracellular matrix. The concentrated repertoire of the proteins in matrix adhesions of myofibroblasts (i.e., over 450 different proteins) and their important role in regulating the metabolic activities of myofibroblasts, has motivated in-depth analysis of their protein complement and how this repertoire is influenced by experimental conditions.In this protocol I describe in detail: (1) the method for isolating focal adhesion-associated proteins using matrix ligand-bound magnetite beads; (2) the method for eluting the proteins from the beads and their preparation for mass spectrometry (Fig. 1). I also briefly consider the mass spectrometry methods including the use of isobaric tags to enable multifactorial experiments and the analysis of the identified proteins. I consider the advantages of these approaches, and the challenges and pitfalls that are encountered with these methods.
Keywords: Extracellular matrix; Fibroblasts; Focal adhesions; Isobaric tags; Mass spectrometry; Signaling.