While the inherent insolubility of many ECM proteins lends itself to effective enrichment by decellularization, subsequent proteomic analysis requires all proteins to be solubilized. Standard lysis buffers are not effective for ECM solubilisation. Instead, we implemented a stepwise extraction of vascular ECM proteins.57 This involves treating vascular tissues with sodium chloride (NaCl) to remove plasma proteins and extract loosely bound extracellular proteins before decellularizing the tissue with sodium dodecyl sulfate (SDS). Each incubation step takes 4 h. Solubilisation of mature ECM proteins is finally achieved by treatment with guanidine hydrochloride (GuHCl) which destabilizes the ionic, disulfide-dependent protein conformations in large aggregating proteoglycans (versican, aggrecan, etc.), small proteoglycans (decorin, biglycan, etc.), cell-attachment glycoproteins such as type VI collagen, fibronectins, laminins, and basement membrane components.67 The method was later adapted for the use in porcine cardiac tissue by reducing the incubation time for NaCl and prolonging the SDS treatment2 (Figure 3A). In smaller animal models (i.e. mouse, rat) cardiac cellularity is proportionally higher compared to that of larger animal models such as pig or goat (Figure 3B). With increased cellularity, decellularization is more difficult to achieve and may require additional enrichment steps, i.e. for glycoproteins or glycopeptides.52 Figure 3Enrichment of cardiac ECM proteins. (A) Our previously published 3-step ECM extraction method for cardiac tissue is based on decellularization and ensures enrichment and detection of ECM proteins. The image shows a decellularized heart after prolonged SDS perfusion. The ECM is solubilized by GuHCl and analysed using proteomics. (B) Smaller species display higher levels of cardiac cellularity as measured by the ratio of 3 members of different ECM protein classes and the cardiac-specific troponin T (TNNT2, y-axis). (C) Proteins identified in murine hearts using the Texas 3-step66 extraction method compared to those identified by our previously published method (see Drozdov et al.61). Most proteins are identified by both methods. Unlike the Texas 3-step method, our ‘English Quickstep’ method did not include an analysis of the remaining pellet after GuHCl extraction.