Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis.
The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology; anaba@mit.edu.
Alexandra
A
Naba
Proteomics Platform, Broad Institute.
Karl R
KR
Clauser
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology.
Richard O
RO
Hynes
D000818:Animals; D049109:Cell Proliferation; D005109:Extracellular Matrix / Q000737:chemistry*; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005355:Fibrosis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D009369:Neoplasms / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification
D016326:Extracellular Matrix Proteins; D010455:Peptides