title: Convenient method to assess chemical modification of protein thiols by electrophilic metals.
abstract: Although covalent modification of protein thiols by electrophilic metals is implicated in disruption of protein functions associated with toxicity, there are limited methods available to detect such modifications. In the present study, we established a convenient method to assess modification of protein thiols by electrophiles, referred to as a biotin-PEAC₅-maleimide (BPM)-labeling assay. In this assay, protein S-modification by electrophiles can be estimated by a decrease in protein modification by BPM, a thiol reactive probe. Using methylmercury (MeHg) as a model electrophilic metal, thiol modification of cellular proteins was detected by the BPM-labeling assay in SH-SY5Y cell lysates and primary mouse hepatocytes. The sensitivity and reliability of the assay was confirmed by atomic absorption spectrometry with recombinant Keap1 as a model thiol protein. This assay was applied to not only MeHg but also to other metals such as cadmium and lead. We also established a BPM-precipitation assay with avidin-agarose beads to separate BPM-modified cellular proteins followed by detection with the individual antibodies. This assay was available for detecting MeHg-induced S-modification of cellular Keap1 in SH-SY5Y cells. Taken together, we have developed reliable simple methods to estimate protein S-modification by electrophilic metals.
authors.affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Toyama
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shinkai
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kaji
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kumagai
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000633:toxicity; D000818:Animals; D001710:Biotin; D003545:Cysteine; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1; D008297:Male; D008301:Maleimides; D008767:Methylmercury Compounds; D051379:Mice; D008810:Mice, Inbred C57BL; D009447:Neuroblastoma / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000633:toxicity; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D008301:Maleimides; D008767:Methylmercury Compounds; D013438:Sulfhydryl Compounds; C043592:maleimide; D001710:Biotin; D003545:Cysteine