Ramallo Guevara Carina, Philipp Oliver, Hamann Andrea, Werner Alexandra, Osiewacz Heinz D, Rexroth Sascha, Rögner Matthias, Poetsch Ansgar
From the ‡Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University Bochum, Bochum-44801, Germany;
§Molecular Developmental Biology, Faculty of Biosciences and Cluster of Excellence 'Macromolecular Complexes', Johann Wolfgang Goethe University, Frankfurt am Main-60438, Germany; ¶Molecular Bioinformatics, Faculty of Computer Science and Mathematics and Cluster of Excellence 'Macromolecular Complexes', Johann Wolfgang Goethe University, Frankfurt am Main-60325, Germany.
Mol Cell Proteomics. 2016 May;15(5):1692-709. doi: 10.1074/mcp.M115.055616. Epub 2016 Feb 16.
The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age.
衰老的自由基理论基于这样一种观点,即活性氧(ROS)可能导致与年龄相关的蛋白质氧化积累。由于细胞内的大多数ROS是在呼吸电子传递链中产生的,本研究聚焦于衰老模型嗜热栖热放线菌的线粒体蛋白质组,将其作为ROS诱导损伤的靶点。为确保检测到低丰度的修饰肽段,开发了通过长梯度nLC-ESI-MS/MS分离和用于iTRAQ定量的适当统计工作流程。通过在存在还原剂和铁螯合剂的情况下建立无凝胶样品制备方法,将人工蛋白质氧化降至最低。这项针对嗜热栖热放线菌的首次大规模、以氧化修饰为中心的研究,实现了对22种不同氧化氨基酸修饰的全面定量,尤其是对氧化和未氧化蛋白质种类的定量比较。总共对2341种蛋白质进行了定量。检测到746种蛋白质的两种形式(未修饰和氧化修饰),并确定了修饰位点。数据显示甲硫氨酸残基更容易被氧化。按出现频率递减顺序进一步显著鉴定出的修饰包括羰基化以及N-甲酰基犬尿氨酸和吡咯烷酮的形成。有趣的是,对于大多数蛋白质,观察到蛋白质含量变化与氧化损伤呈正相关,并且在衰老后期蛋白质含量普遍下降。然而,发现少数蛋白质的氧化损伤变化与先前报道一致。我们的数据表明,只要蛋白质从头合成功能正常,嗜热栖热放线菌在衰老过程中能够有效地抵抗ROS诱导的蛋白质损伤,最终导致受损和未受损蛋白质种类之间的总体恒定关系。这些发现与衰老过程中蛋白质氧化的大量增加相矛盾,反而表明即使在衰老后期也存在蛋白质损伤稳态机制。