Isolation and Proteomic Analysis of Valvular Interstitial Cells

VICs were isolated from AVs removed for severe stenosis using tissue lysis with 2% collagenase (Sigma-Aldrich, St Louis, MO, USA). Protein extraction and proteolysis were performed with the methanol-chloroform method and trypsin [(Gold Grade; Promega, Wisconsin)/RapiGest procedure (Waters, Milford, MA, USA)], respectively, as previously published (18 (link)). Fifteen micrograms of protein was used per sample. The tryptic peptides were desalted using Oasis Hlb 1cc (10 mg) columns (Waters, Milford, MA, USA), and dried with a tabletop speed vacuum (SPD1010, Thermo Fisher Scientific, USA). After resuspension in 40 μl of 5% mass spectrometry grade acetonitrile (Thermo Fisher Scientific, USA) and 5% formic acid (Sigma-Aldrich, St Louis, MO, USA), the tryptic peptide samples were analyzed by liquid chromatography–mass spectrometry.