Enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis

Abstract

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are majorregulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies includingcardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseasedtissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, thevery nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses ofECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takesadvantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubationsin buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membraneand cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched proteinpreparations into peptides for subsequent analysis by mass spectrometry.