Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada.
PhenoSwitch Bioscience, 975 Rue Léon-Trépanier, Sherbrooke, Quebec, J1G 5J6, Canada.
Rapid Commun Mass Spectrom. 2020 Jun 15;34(11):e8774. doi: 10.1002/rcm.8774.
Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry.
Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS.
ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile.
Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.
氧化应激是活性氧自由基与抗氧化防御之间失衡的结果。其后果可导致多种病理。调节氧化应激是抗氧化剂再循环和含硫调节蛋白之间复杂的相互作用。了解这些调节机制对于预防氧化应激的发生很重要。本研究旨在使用质谱法研究与氧化维生素 C（脱氢抗坏血酸，DHA）、同型半胱氨酸（HcySH）和谷胱甘肽（GSH）相关的 S-巯基蛋白化学。
用 DHA、有和没有 GSH 和 HcySH 孵育谷胱甘肽还原酶-1（Grx-1）。通过电喷雾电离质谱（ESI-MS）对完整蛋白质进行分析，并通过蛋白质胰酶消化肽的 LC/ESI-MS/MS 分析来跟踪二硫键的形成。使用两种合成肽 AcFHACAAK 和 AcFHACE 研究 DHA 介导的 S-巯基化的机制。三种蛋白质，即人血红蛋白（HHb）、重组过氧化物酶 2（Prdx2）和 Grx-1，被 S-同型半胱氨酸化，然后用 GSH 进行 S-转硫醇化，并通过 ESI-MS 和 ESI-MS/MS 进行研究。
ESI-MS 分析表明，DHA 通过 HcySH 以及 GSH 介导 Grx-1 的二硫键形成和 S-巯基化。LC/ESI-MS/MS 分析允许鉴定 Grx-1 的 S-巯基化半胱氨酸加合物。DHA 介导七肽 AcFHACAAK 的 S-巯基化的机制被证明是通过初始形成硫代半缩酮加合物。此外，完整蛋白质的 ESI-MS 显示 GSH 可以 S-转硫醇化 S-同型半胱氨酸化的 Grx-1_HHb 和 Prdx2。GS-S-蛋白质加合物随时间推移占据 ESI-MS 光谱谱图的主导地位。
质谱是一种独特的分析技术，可用于探测与氧化应激相关的复杂反应机制。使用模型蛋白质，ESI-MS 揭示了 DHA 促进的 S-巯基化的机制，该机制包括硫代半缩酮形成、二硫键形成或 S-巯基化。此外，HcySH 引起的蛋白质 S-巯基化可以通过可逆的 GSH 硫醇交换逆转。在氧化应激中使用蛋白质 S-巯基化的体外模型与质谱联用，可能为体内发生的可能作用机制提供重要的见解。
