title: Detection of protein adduction derived from dauricine by alkaline permethylation.
abstract: 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.
authors.affiliation: Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China.
authors.forename: Honglei
authors.initials: H
authors.lastname: Xie
authors.affiliation: School of Pharmacy Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China.
authors.forename: Yuyang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: School of Pharmacy Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China. yingpeng1999@163.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China. dongmeiz-67@163.com.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China. zhengneu@yahoo.com.|Center for Developmental Therapeutics, Seattle Children's Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, 98101, USA. zhengneu@yahoo.com.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zheng
mesh_terms: D000468:Alkalies / Q000737:chemistry*; D044182:Benzylisoquinolines / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D008745:Methylation; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D044005:Tetrahydroisoquinolines / Q000737:chemistry*
chemical_list: D000468:Alkalies; D044182:Benzylisoquinolines; D011506:Proteins; D044005:Tetrahydroisoquinolines; C035934:dauricine
keywords: Alkaline permethylation; Dauricine; Protein adduction