Mass spectrometry was used to investigate the effects of exposing mitochondrial aconitase (ACO2) to the membrane lipid peroxidation product, 4-hydroxy-2-(E)-nonenal (HNE). ACO2 was selected for this study because (1) it is known to be inactivated by HNE, (2) elevated concentrations of HNE-adducted ACO2 have been associated with disease states, (3) extensive structural information is available, and (4) the iron-sulfur cluster in ACO2 offers a critical target for HNE adduction. The aim of this study was to relate the inactivation of ACO2 by HNE to structural features. Initially, Western blotting and an enzyme activity assay were used to assess aggregate effects and then gel electrophoresis, in-gel digestion, and tandem mass spectrometry (MS/MS) were used to identify HNE addition sites. HNE addition reaction rates were determined for the most significant sites using the iTRAQ approach. The most reactive sites were Cys(358), Cys(421), and Cys(424), the three iron-sulfur cluster-coordinating cysteines, Cys(99), the closest non-ligated cysteine to the cluster, and Cys(565), which is located in the cleft leading to the active site. Interestingly, both enzyme activity assay and iTRAQ relative abundance plots appeared to be trending toward horizontal asymptotes, rather than completion.
Copyright © 2013 Elsevier B.V. All rights reserved.