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 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.

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival

“…1A). To systematically characterize the matrisome of normal lung, fibrotic lung, primary lung adenocarcinoma, and associated lymph node metastases, we used a recently developed proteomics-based approach (9,13). The four sample types-normal lungs, fibrotic lungs, primary tumors, and metastatic tumors-were first subjected to a decellularization procedure to remove the soluble intracellular proteins and to enrich for ECM proteins (Fig.…”

“…Decellularization of samples ranging from 50 to 100 mg was performed using the CNMCS compartmental protein extraction kit (EMD Millipore), as described previously (9,13). In brief, frozen samples were homogenized with a Bullet Blender (Next Advance) according to the manufacturer's instructions and then incubated in a series of buffers to remove, sequentially, cytosolic proteins, nuclear proteins, membrane proteins, and cytoskeletal proteins.…”

The extracellular matrix: Tools and insights for the “omics” era

“…Taking advantage of the insolubility of ECM proteins, we and others have reported the development of decellularization and ECM-enrichment methods that rely on the extraction of - somewhat more soluble - intracellular proteins [16, 43–46]. The insolubility of ECM proteins is, however, a challenge for subsequent proteomic analyses; indeed mass-spectrometry pipelines require proteins to be solubilized and then digested into peptides.…”

“…Therefore, we proposed that crude ECM-enriched samples be denatured, reduced and alkylated, deglycosylated, and digested by a combination of proteases (LysC and trypsin), all without intervening centrifugation, in order to minimize losses of otherwise insoluble materials. Using this approach, we could show that solubilization occurs as a concomitant of protease treatment [16, 43]. …”

“…Matrisome-associated proteins are also likely to be more soluble than core matrisome proteins and could be lost during decellularization. One approach to retrieve information on more soluble proteins would be to profile not only the composition of the insoluble ECM fraction generated by tissue decellularization but also the composition of the intermediate fractions generated during the decellularization process [43, 63] and the soluble components of tissue interstitial fluid. Of course, these intermediate fractions will also be enriched for intracellular components, thus robust data annotations using matrisome lists could assist with delineating which proteins are soluble matrisome proteins and which are contaminants.…”