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Identification of creatine kinase and alpha‐1 antitrypsin as protein targets of alkylation by sulfur mustard
Drug Testing and Analysis ( IF 2.9 ) Pub Date : 2020-08-27 , DOI: 10.1002/dta.2916
Robin Lüling 1, 2 , Wolfgang Schmeißer 1 , Markus Siegert 1, 3 , Harald Mückter 2 , Alexander Dietrich 2 , Horst Thiermann 1 , Thomas Gudermann 2 , Harald John 1 , Dirk Steinritz 1, 2, 4
Affiliation  

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two‐dimensional‐thiol‐differences in gel electrophoresis (2D‐thiol‐DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared‐maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide–thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D‐thiol‐DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix‐assisted laser desorption/ionization time‐of‐flight (tandem) mass spectrometry, MALDI‐TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha‐1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys282 of CK and Cys232 of A1AT were unambiguously identified by micro liquid chromatography‐electrospray ionization high‐resolution tandem‐mass spectrometry (μLC‐ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl‐(HETE)‐moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.

中文翻译:

鉴定肌酸激酶和 α-1 抗胰蛋白酶作为硫芥烷化的蛋白质靶标

硫芥 (SM) 是一种有毒化学战剂,在过去 100 年间曾部署在数次冲突中,并且在恐怖袭击和战争中仍然构成威胁。SM 研究侧重于了解 SM 的病理生理学和识别新的暴露生物标志物。已知 SM 将内源性蛋白质的亲核部分烷基化,例如,半胱氨酸残基的游离硫醇基团。凝胶电泳中的二维硫醇差异 (2D-thiol-DIGE) 技术是检测具有游离半胱氨酸残基的蛋白质的初始蛋白质组学方法。这些氨基酸被 GE 后可见的红外马来酰亚胺染料选择性标记。由烷化剂衍生的半胱氨酸残基不再可用于马来酰亚胺-硫醇偶联,导致相应蛋白质的荧光信号丢失。为了证明 2D-硫醇-DIGE 的适用性,该技术被示例性地应用于用 SM 处理的纯人血清白蛋白、来自暴露于 SM 的人类细胞培养物以及暴露于 CEES(氯乙基乙基硫醚,一种 SM类似物)。例如,通过基质辅助激光解吸/电离飞行时间(串联)质谱法、MALDI-TOF MS(/MS) 鉴定了经 SM 修饰的最突出的蛋白质,作为来自人体细胞的肌酸激酶 (CK) 和作为来自血浆样本的 alpha-1 抗胰蛋白酶 (A1AT)。含有残基 Cys 的肽 通过基质辅助激光解吸/电离飞行时间(串联)质谱法、MALDI-TOF MS(/MS) 鉴定出由 SM 修饰的最突出的蛋白质,如来自人体细胞的肌酸激酶 (CK) 和 α- 1 来自血浆样品的抗胰蛋白酶 (A1AT)。含有残基 Cys 的肽 通过基质辅助激光解吸/电离飞行时间(串联)质谱法、MALDI-TOF MS(/MS) 鉴定出由 SM 修饰的最突出的蛋白质,如来自人体细胞的肌酸激酶 (CK) 和 α- 1 来自血浆样品的抗胰蛋白酶 (A1AT)。含有残基 Cys 的肽A1AT的 CK 的282和 Cys 的232被微液相色谱-电喷雾电离高分辨率串联质谱 (μLC-ESI MS/HR MS) 明确鉴定为被带有特定羟乙基硫乙基-(HETE)-部分的 SM 烷基化。两种肽都可能代表 SM 暴露的潜在生物标志物。这是第一份将这些内源性蛋白质作为 SM 烷基化目标的报告。
更新日期:2020-08-27
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