Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428.