Peptide Identification from ECM Matrices

For peptide identification, soluble and insoluble fractions of HMVEC and HDF matrices digested with Santyl^® collagenase were collected into ECM-solubilization buffer containing 20 mM Tris-Cl (pH 8.0), 150 mM NaCl (pH 7.0), 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% NP-40, supplemented with protease inhibitors (P8340, Sigma-Aldrich). The digested matrices were then mixed with reducing sample buffer containing a final concentration of 2% β-mercaptoethanol (Sigma-Aldrich), heated to 95°C for 5 minutes, separated by SDS-PAGE, and proteins were stained using the SilverQuest Silver Staining Kit (ThermoFisher Scientific, Waltham, MA). Protein bands of interest were excised from the gels, and submitted to Taplin Mass Spectrometry Facility at Harvard Medical School for protein identification. At Taplin, the gel bands were degraded using proteomics-grade trypsin, and subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using Orbitrap mass spectrometers (ThermoFisher Scientific). Over 100 protein fragments were identified in Santyl^®-digested matrices; of these, 8 peptides derived from HMVEC matrix and 6 peptides derived from HDF matrix, each containing 12–25 amino acids, were selected and submitted to Tufts University Core Facility (TUCF) for synthesis by FastMoc Chemistry, as previously described [26 (link),30 ,31 (link)].