Xie Honglei, Liu Yuyang, Peng Ying, Zhao Dongmei, Zheng Jiang
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China.
School of Pharmacy Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China.
Anal Bioanal Chem. 2016 Jun;408(15):4111-9. doi: 10.1007/s00216-016-9505-0. Epub 2016 Apr 12.
Dauricine is a bisbenzylisoquinoline alkaloid derivative and has shown multiple pharmacological properties. Despite this, our previous study demonstrated that dauricine induced severe lung toxicity in experimental animals. Metabolic activation of dauricine to the corresponding quinone methide intermediate is suggested to play an important role in dauricine-induced cytotoxicity. Protein adduction derived from the reactive intermediate is considered to initiate the process of the toxicity. In the present study, we developed an alkaline permethylation- and mass spectrometry-based approach to detect dauricine-derived protein adduction. Protein samples were permethylated in the presence of NaOH and CH3I at 80 °C, followed by LC-MS/MS analysis. A thioether product was produced in the reaction. Not only does this technique quantify dauricine-derived protein adduction but also it tells the nature of the interaction between the target proteins and the reactive intermediate of dauricine. The recovery, precision, limit of detection, limit of quantity, and method detection limit were found to be 102.8 %±1.7 %, 1.89 %, 1.32 fmol/mL, 4.93 fmol/mL and 3.37 fmol/mL respectively. The surrogate recovery and surrogate RSD values were 81.5-103.0 % and 2.59 %, respectively. This analytical method has proven sensitive, selective, reliable, and feasible to assess total protein adduction derived from dauricine, and will facilitate the mechanistic investigation of dauricine and other bisbenzylisoquinoline toxicities. Graphical Abstract Alkaline permethylation of dauricine derived protein adduct.
蝙蝠葛碱是一种双苄基异喹啉生物碱衍生物，具有多种药理特性。尽管如此，我们之前的研究表明，蝙蝠葛碱在实验动物中会引发严重的肺毒性。蝙蝠葛碱代谢活化生成相应的醌甲基化物中间体被认为在其诱导的细胞毒性中起重要作用。由反应性中间体衍生的蛋白质加合物被认为启动了毒性过程。在本研究中，我们开发了一种基于碱性甲基化和质谱的方法来检测蝙蝠葛碱衍生的蛋白质加合物。蛋白质样品在NaOH和CH3I存在下于80°C进行甲基化，随后进行LC-MS/MS分析。反应中产生了一种硫醚产物。该技术不仅能定量蝙蝠葛碱衍生的蛋白质加合物，还能揭示目标蛋白与蝙蝠葛碱反应性中间体之间相互作用的性质。回收率、精密度、检测限、定量限和方法检测限分别为102.8%±1.7%、1.89%、1.32 fmol/mL、4.93 fmol/mL和3.37 fmol/mL。替代回收率和替代RSD值分别为81.5 - 103.0%和2.59%。这种分析方法已被证明对评估蝙蝠葛碱衍生的总蛋白质加合物敏感、选择性好、可靠且可行，将有助于对蝙蝠葛碱和其他双苄基异喹啉毒性的作用机制进行研究。图形摘要：蝙蝠葛碱衍生蛋白质加合物的碱性甲基化。
