1 research outputs found
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