Institut des Sciences Analytiques, UMR, 5280, Université Lyon 1, CNRS, Villeurbanne, France.
Laboratoire de Chimie ENS Lyon, UMR, 5582, ENS Lyon CNRS et Université Lyon 1, France.
Talanta. 2022 Dec 1;250:123745. doi: 10.1016/j.talanta.2022.123745. Epub 2022 Jul 19.
Cysteine (Cys) is subject to a variety of reversible post-translational modifications such as formation of sulfenic acid (Cys-SOH). If this modification is often involved in normal biological activities, it can also be the result of oxidative damage. Indeed, oxidative stress yields abnormal cysteine oxidations that affect protein function and structure and can lead to neurodegenerative diseases. In a context of population ageing, validation of novel biomarkers for detection of neurodegenerative diseases is important. However, Cys-SOH proteins investigation in large human cohorts is challenging due to their low abundance and lability under endogenous conditions. To improve the detection specificity towards the oxidized protein subpopulation, we developed a method that makes use of a mass spectrometer coupled with visible laser induced dissociation (LID) to add a stringent optical specificity to the mass selectivity. Since peptides do not naturally absorb in the visible range, this approach relies on the proper chemical derivatization of Cys-SOH with a chromophore functionalized with a cyclohexanedione. To compensate for the significant variability in total protein expression within the samples and any experimental bias, a normalizing strategy using free thiol (Cys-SH) cysteine peptides derivatized with a maleimide chromophore as internal references was used. Thanks to the differential tagging, oxidative ratios were then obtained for 69 Cys-containing peptides from 19 proteins tracked by parallel reaction monitoring (PRM) LID, in a cohort of 49 human plasma samples from Alzheimer disease (AD) patients. A statistical analysis indicated that, for the proteins monitored, the Cys oxidative ratio does not correlate with the diagnosis of AD. Nevertheless, the PRM-LID method allows the unbiased, sensitive and robust relative quantification of Cys oxidation within cohorts of samples.
半胱氨酸（Cys）可发生多种可逆的翻译后修饰，如亚磺酸（Cys-SOH）的形成。如果这种修饰经常参与正常的生物活性，也可能是氧化损伤的结果。事实上，氧化应激会导致异常的半胱氨酸氧化，从而影响蛋白质的功能和结构，并导致神经退行性疾病。在人口老龄化的背景下，验证用于检测神经退行性疾病的新型生物标志物非常重要。然而，由于内源性条件下 Cys-SOH 蛋白的丰度低且不稳定性，在大型人类队列中研究 Cys-SOH 蛋白具有挑战性。为了提高对氧化蛋白亚群的检测特异性，我们开发了一种方法，该方法利用质谱仪与可见激光诱导解离（LID）相结合，为质量选择性增加严格的光学特异性。由于肽在可见光范围内不会自然吸收，因此该方法依赖于用功能化有环己二酮的生色团对 Cys-SOH 进行适当的化学衍生化。为了补偿样品中总蛋白表达的显著变化和任何实验偏差，使用了一种归一化策略，该策略使用用马来酰亚胺生色团衍生化的自由巯基（Cys-SH）半胱氨酸肽作为内部参考。通过差异标记，然后在 AD 患者的 49 个人血浆样本的队列中，使用平行反应监测（PRM）LID 跟踪 19 个蛋白质的 69 个含半胱氨酸的肽获得了氧化比值。统计分析表明，对于监测的蛋白质，Cys 氧化比值与 AD 的诊断不相关。然而，PRM-LID 方法允许在样本队列中进行无偏、敏感和稳健的 Cys 氧化相对定量。
