Comparative Profiling of the Mammalian Mitochondrial Proteome:  Multiple Aconitase-2 Isoforms Including N-formylkynurenine Modifications as Part of a Protein Biomarker Signature for Reactive Oxidative Species

Abstract

The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

Mitochondrial proteome analysis reveals altered expression of voltage dependent anion channels in pancreatic β-cells exposed to high glucose

“…11 Different isoforms of aconitase have been detected in 2-D protein map 26 and the activity of aconitase has been found be sensitive to oxidative conditions. 27 During oxidative stress and altered metabolic conditions aconitase relocates from the TCA cycle to mtDNA nucleoids, where it stabilizes the genome under these adverse conditions.…”

Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria

“…Each lane was loaded with the extract from mitochondria containing 150 g (for in-gel activity), 300 g (for 2D SDS-PAGE), or 400 g (for 2D BN-PAGE) of protein before solubilization which was assessed by Bradford assay. For BN-PAGE and CN-PAGE, linear 3 to 13% gradient gels overlaid with a 3% stacking gel were used in a Hoefer SE 600 system (18 by 16 by 0.15 cm 3 ) (40,69,72). CN-PAGE was performed according to the methods of Schägger et al (72), omitting Coomassie blue dye in the cathode buffer and without the addition of detergent in the gel (46,61).…”

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

“…The combined application of conventional 2-DE with denaturing/nondenaturing, unconventional 2-DE approaches has been suggested to be sufficient to provide a repertory of all membrane proteins of a given sample [144,164].…”