Fig. 2.

Proteomics workflow. The methodology is based on a three-step subfractionation of the vascular proteome (A), which enriches ECM, its associated proteins and degradation products. First, specimens are extracted in 0.5 m NaCl. This buffer solubilizes proteins or protein fragments that are weakly bound to the ECM, including newly synthesized ECM proteins and degradation products. In addition, secreted proteinases such as MMPs are also extracted. Second, specimens are decellularized with 0.08% SDS to deplete cellular proteins. Finally, the salt-insoluble ECM proteins are extracted using 4 m guanidine to solubilize the tightly interacting, cross-linked matrix. In the second stage (B), the identified MMPs are further investigated by incubating healthy tissue with recombinant enzymes to identify novel proteolytic targets and characterize their proteolytic activity in disease.