Proteome differences in cardiomyocytes from hCOX-2-Tg mice

[Methods] Peptides and proteins from cardiomyocytes were trypsin-digested using the whole proteome in-gel digestion protocol, followed by 18O labeling as previously described (Bonzon-Kulichenko, E. et al Mol. Cell. Proteomics 2011, 10, M110 003335, doi:10.1074/mcp.M110.003335). The peptide pools were separated in 24 fractions ranging from pH 3-10 by IEF on a 3100 OFFGel fractionator (Agilent, Santa Clara, CA, USA) using the standard methods for peptides recommended by the manufacturer. The recovered fractions were desalted using OMIX C18 tips (Varian, Inc, Agilent, USA), and dried down before reverse phase-high performance liquid chromatography (RP-HPLC)-LIT analysis using a Surveyor LC System coupled to a linear ion trap mass spectrometer LTQ (Thermo Fisher Scientific, Waltham, MA USA). The LTQ was operated in a data-dependent ZoomScan and MS/MS switching mode as previously described (Lopez-Ferrer, D. et al. Proteomics 2006, 6 Suppl 1, S4-11, doi:10.1002/pmic.200500375). Protein identification was done using SEQUEST algorithm (Bioworks 3.2 package, Thermo Fisher Scientific). MS/MS raw files were searched against a Rat/Mouse Swissprot database supplemented with the sequence of bovine and porcine trypsin. SEQUEST results were analyzed using the probability ratio method (Martinez-Bartolome, S et al. Mol. Cell. Proteomics 2008, 7, 1135–1145, doi:10.1074/mcp.M700239-MCP200) and discovery rates (FDR) of peptide identifications were calculated as previously described ((Navarro, P. et al. J Proteome Res 2009, 8, 1792–1796, doi:10.1021/pr800362h). Peptide identification and quantification were done as previously described (Bonzon-Kulichenko, E. et al Mol. Cell. Proteomics 2011, 10, M110 003335, doi:10.1074/mcp.M110.003335; Navarro, P.; et al. J Proteome Res 2014, 13, 1234–1247, doi:10.1021/pr4006958). Statistical significance of protein abundance changes was assayed by controling the FDR, being a FDR less than 0.05 considered to be significant. Threshold-free analysis of coordinated protein responses was performed using the SBT model, as described (García-Marqués, F. et al. Mol. Cell. Proteomics 2016, 15, 1740–1760, doi:10.1074/mcp.M115.055905).