BUSCADOR RECOLECTA

Digital.CSIC. Repositorio Institucional del CSIC

oai:digital.csic.es:10261/280146

[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)., The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice, thatconstitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no an apparent phenotype, but were protected against ischemia-reperfusion injury in isolated hearts, with an enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. Here we include the results of this proteomic study with the list of identified proteins and their quantification, Ministerio de Economia y Competitividad (SAF2013-43713-R) and Generalitat Valenciana (ACOMP/2011/120), No