Gu Liqing, Robinson Renã A S
Department of Chemistry, University of Pittsburgh, 111 Eberly Hall, 200 University Drive, Pittsburgh, PA, 15260, USA.
Anal Bioanal Chem. 2016 Apr;408(11):2993-3004. doi: 10.1007/s00216-016-9307-4. Epub 2016 Jan 22.
Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD.
半胱氨酸通过多种可逆和不可逆修饰广泛参与氧化还原信号通路。可逆修饰（如S-谷胱甘肽化、S-亚硝基化、二硫键和亚磺酸）用于保护蛋白质免受氧化攻击并维持细胞内稳态，而不可逆氧化（如亚磺酸和磺酸）则是氧化应激的标志。对富含半胱氨酸的肽段进行蛋白质组学分析并结合氧化硫醇的还原，可用于测量半胱氨酸的氧化状态，这有助于阐明氧化应激在生物学和疾病中所起的作用。作为我们之前报道的cysDML方法的扩展，我们开发了氧化半胱氨酸选择性二甲基化（OxcysDML）方法，以研究生物学相关样品中半胱氨酸残基的位点特异性总氧化情况。OxcysDML方法包括：（1）用半胱氨酸反应试剂封闭游离硫醇；（2）用固相树脂富集含有可逆氧化半胱氨酸的肽段；（3）对肽段氨基进行同位素标记，以量化不同生物学条件下产生的半胱氨酸修饰。与其他氧化还原蛋白质组学方法相比，树脂上富集和标记可最大限度减少样品处理时间并提高效率。OxcysDML方法还具有成本低且灵活的特点，因为它可以用于探索各种半胱氨酸修饰。在此，我们将该方法应用于晚期阿尔茨海默病（AD）小鼠模型和野生型（WT）对照的肝脏组织。由于我们之前已使用cysDML方法对该蛋白质组进行过表征，因此我们在此能够更深入地探究AD中半胱氨酸的氧化还原状态。OxcysDML方法鉴定出1129个半胱氨酸位点（来自527种蛋白质），其中828个半胱氨酸位点发生了氧化修饰。19个氧化半胱氨酸位点在AD中具有显著的变化水平，且代表参与代谢过程的蛋白质。总体而言，我们已证明OxcysDML是一种简单、快速、稳健且廉价的氧化还原蛋白质组学方法，有助于更深入地了解AD的蛋白质组。
