Desarrollo de nuevas tecnologías en proteómica de segunda generación y su aplicación al estudio de cambios dinámicos relacionados con la disfunción cardiovascular

Abstract

In spite of recent developments, the global analysis of membrane subproteomes is still a common obstacle. Also, in some situations, the precise characterization of a particular peptide species present in low amounts in a very complex peptide mixture is needed. In this work we have developed novel proteomic approaches for high sensitivity analysis of specific peptides of membrane proteins in complex samples, as well as for high-throughput membrane protein identification and quantification. Also, we show some applications of these techniques in the context of biological studies on cardiovascular dysfunction. Ischemic preconditioning protects against ischemia-reperfusion injury by still unknown molecular mechanisms. Mitochondria have been proposed to be involved in cardioprotection exherted by ischemic preconditioning. In this study, differential protein expression in myocardial mitochondria was assessed during rat ischemic preconditioning by high-throughput proteomics. 300 µg of SDS-solubilized mitochondria from the hearts of normoxic and preconditioned rats were separated on SDS-PAGE. The gel was cut into several fractions, which were subjected to trypsin digestion, followed by stable isotope labeling with 16O/18O and RP-HPLC-LIT analysis. In-house developed programs pRatio and QuiXoT were used for high-throughput peptide identification and quantification, respectively. This analysis resulted in confident identification of more than 400 mitochondrial proteins, from which a large proportion could be quantified. The abundance of several mitochondrial membrane proteins was statistically increased or decreased, being most of them involved in energy metabolism and mitochondrial dysfunction. The set of differentially expressed proteins is consistent with a protective role in miocardium of preconditioned rats. Connexin 43 plays an important role in cardiac preconditioning. However, the presence of this protein in mitochondria has never been described in a proteomic study. Here we verified the presence of Cx43 in purified mitochondrial preparations from mouse myocardium by a mass-spectrometry-based proteomic approach, Also, we demonstrate the absence of Cx32 in cardiac mitochondria and its presence in hepatic mitochondria in preconditioning-resistant Cx43KICx32 mice. Our results may help understanding the molecular mechanisms involved in ischemia preconditioning.