Detection of cysteine- and lysine-based protein adductions by reactive metabolites of 2,5-dimethylfuran.
Many furan-containing compounds are known to be toxic and/or carcinogenic. Metabolic activation of toxic furans to cis-enediones (cis-enedials or γ-ketoenals) is generally considered as the initial step towards the processes of their toxicities. Sequential modification of key proteins by the electrophilic reactive intermediates is suggested to be an important mechanism of the toxic actions. In the present study, we developed a novel and simple analytical platform to detect protein modification resulting from metabolic activation of model compound 2,5-dimethylfuran (DMF). 4-Bromobenzylamine and 4-bromobenzylmercaptan were employed to trap protein adductions at cysteine and lysine residues, respectively. The resulting protein samples were proteolytically digested by chymotrypsin and Pronase E, followed by LC-MS/MS analysis. Modifications of cysteine and lysine residues of proteins were observed in microsomal incubations and animals after exposure to DMF. In conclusion, the approach established has been proven highly selective and reliable. This advance allows us not only to detect the protein adductions but also to define the structural identities of amino acid residues modified. This technique provides a unique platform to assess protein modifications arising from metabolic activation of potentially harmful furan-containing compounds. Hepatic protein adductions were found to be proportional to the hepatotoxicity of DMF.
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
Kai
K
Wang
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China.
Weiwei
W
Li
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
Jiaming
J
Chen
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China. Electronic address: yingpeng1999@163.com.
Ying
Y
Peng
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; 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. Electronic address: jiang.zheng@seattlechildrens.org.
Jiang
J
Zheng
D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005663:Furans / Q000737:chemistry* / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D005663:Furans; D011506:Proteins; C037555:2,5-dimethylfuran; D008239:Lysine; D003545:Cysteine
2,5-Dimethylfuran; Bromine-tagged reagents; Furan-containing compounds; Protein adductions; Reactive metabolite