A Multi-enzyme Protocol Improves Total Proteome Coverage in Extracellular Matrix

Extracellular matrix (ECM) from decellularized mammalian tissues has been used in many therapeutic applications. The tissue-specific composition of the ECM is critically associated with therapeutic performance. However, ECM translation needs to be improved because of the complex composition and limited understanding of ECM repairing mechanisms due partly to incomplete proteomic interrogation of ECM samples. In this chapter, we describe a multi-enzyme, bottom-up proteomics workflow employing trypsin, Lys-C, collagenase, and elastase to enhance the digestion of ECM and increase total protein coverage. The outcomes from the reported approach, in a standardized manner, enable users to pinpoint changes in the ECM composition, thereby facilitating the establishment of mechanistic correlations between ECM composition and its effects.