Araki Kazutaka, Kusano Hidewo, Sasaki Naoyuki, Tanaka Riko, Hatta Tomohisa, Fukui Kazuhiko, Natsume Tohru
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology , Tokyo 135-0064, Japan.
Robotic Biology Institute, Inc. , Tokyo 135-0064, Japan.
J Proteome Res. 2016 Aug 5;15(8):2548-59. doi: 10.1021/acs.jproteome.6b00087. Epub 2016 Jul 15.
The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2), uroporphyrinogen decarboxylase (UROD), and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 μM for 5 min) did not cause global oxidations but instead had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.
蛋白质半胱氨酸残基是最易受到氧化修饰的氨基酸之一，这种修饰常由氧化应激引起。有几种方法可用于靶向半胱氨酸的蛋白质组学分析，并能同时进行检测和定量。在本研究中，我们采用了一种定量方法，使用一组基于碘乙酰基的半胱氨酸反应性等压标签（碘TMT），并评估了在二硫苏糖醇（DTT）和过氧化氢（H2O2）处理下，游离和可逆修饰的半胱氨酸硫醇的瞬时细胞氧化率。DTT处理（1 mM，5分钟）使大多数半胱氨酸硫醇还原，无论其细胞定位如何。它还引起了一些独特的氧化变化，包括过氧化物酶2（PRDX2）、尿卟啉原脱羧酶（UROD）和硫氧还蛋白（TXN），据报道这些蛋白质会受到细胞活性氧产生的影响。适度的H2O2处理（50 μM，5分钟）不会引起全局氧化，反而具有明显的还原作用。此外，在H2O2处理下，仅在氧化还原活性蛋白中观察到显著的氧化变化，如PRDX2、过氧化物酶1（PRDX1）、TXN和甘油醛-3-磷酸脱氢酶（GAPDH）。总体而言，我们的定量数据说明了H2O2介导和还原介导的细胞反应，即在维持氧化还原稳态的同时，高反应性硫醇可以增强特定的、快速的细胞信号传导，以对抗急性氧化还原应激。
