These effects can be attributed to presence of gelatin, which can enhance the binding of ECM to the plates preventing the matrix from being dislodged during decellularization. Clomifene citrate However, LC-MS analysis of pure gelatin revealed the presence of several matricellular proteins and hence the presence of gelatin in decellularized matrix during Toremifene Citrate proteomic analysis of cardiogel might lead to unreliable results. Therefore, cardiogel from non-coated plates were used for proteomic analysis, while cardiogel from gelatin coated plates were used in studies evaluating its biological properties such as cytocompatibility and regenerative potential. Fibrous proteins such as collagen and fibronectin are cross-linked in ECM and usually insoluble during protein extraction. A ECM solubilization process, which included treatment with 5% acetic acid followed by buffer containing SDS and DTT, was used to facilitate the solubilization of these matrix proteins. However, the presence of detergents such as SDS can interfere during proteomic analysis and therefore such detergents were removed using acetone precipitation. Finally, surfactant-assisted solubilization of the protein pellet using 0.1% Rapigest SF surfactant was carried out to ensure complete solubilization of cardiogel. The same extraction protocol was followed for extraction of mesogel, which was used as control ECM for cardiogel in proteomic analysis. Preliminary characterization showed that the levels of Collagen I and III, Laminin and Fibronectin were relatively high in cardiogel as compared to mesogel. This difference in the proportion of ECM structural proteins may contribute to the observed biological properties in cardiogel. An in-depth comparison of cardiogel and mesogel using label-free nLC-MS/MS followed by functional clustering using DAVID, revealed that proteins involved in biological processes such as cardiac muscle development, angiogenesis and response to oxidative stress/hydrogen peroxide were unique to cardiogel while those involved in osteogenesis and muscle development were specific to mesogel.