Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection.

The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart. We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation. We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts. We found that phosphorylation of a 46-kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts. Using 2D gel electrophoresis and mass spectrometry, we have identified the 46-kDa protein as mitochondrial translational elongation factor Tu (EF-Tu(mt)). These data reveal that ischemia and preconditioning modulate the phosphorylation of EF-Tu(mt) and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation. Phosphorylation of mitochondrial EF-Tu has not been previously described; however, in prokaryotes, EF-Tu phosphorylation inhibits protein translation. We hypothesized that phosphorylation of mitochondrial EF-Tu would inhibit mitochondrial protein translation and attempted to reproduce the effect with inhibition of mitochondrial protein synthesis by chloramphenicol. We found that chloramphenicol pretreatment significantly reduced infarct size, suggesting that mitochondrial protein synthesis is one determinant of myocardial injury during ischemia and reperfusion.

Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometry.

Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO2-). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3-nitrotyrosine. In this work, we show that mass spectral patterns associated with 3-nitrotyrosine containing peptides allow identification of peptides containing this modification. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(m-NO2)AR and several peptides containing 3-nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of ions were found for these peptides in addition to the mass shift of +45 Da corresponding to the addition of the nitro group. Specifically, two additional ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that suggest the formation of hydroxylamine and amine derivatives. These latter four components appear to originate by laser-induced photochemical decomposition. MALDI-MS analysis of the synthetic peptide containing 3-nitrotyrosine revealed this same pattern. Post-source decay (PSD) MALDI-time-of-flight (TOF) and collisional activation using a prototype MALDI quadrupole TOF yielded extensive fragmentation that allowed site-specific identification of 3-nitrotyrosine. Conversion of peptides containing 3-nitrotyrosine to 3-aminotyrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abundant sidechain loss under PSD conditions. These observations suggest that MALDI can provide a selective method for the analysis and characterization of 3-nitrotyrosine-containing peptides.

Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y neuroblastoma cell line.

To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.