Comparative methods for analysis of protein covalent modification by electrophilic quinoids formed from xenobiotics

Conjugation of biotin and fluorophore tags is useful for assaying covalent protein modification. Oxidative bioactivation of selective estrogen receptor modulators (SERMs) yields reactive quinoid electrophiles that covalently modify proteins, and bioactivation is associated with carcinogenic and chemopreventive effects. Identification of the protein targets of electrophilic metabolites is of general importance for xenobiotics. Four methodologies using SERM derivatized biotin/fluorophore tags were compared for purification and quantification: (1) covert oxidatively activated tags (COATags; SERM conjugated to biotin); (2) dansylTags (SERM conjugated to fluorophore); and azidoTags (SERM azide derivatives) in a two-step conjugation to biotin, using either (3) Staudinger ligation or (4) click chemistry. All synthetic derivatives retained the estrogen receptor ligand characteristics of the parent SERMs. Model proteins with bioactivation by tyrosinase in buffer or cell lysates and liver proteins with in situ bioactivation in rat primary hepatocytes were studied by immunoassay and fluorescence. Comparison showed that the azidoTag/Staudinger method was sensitive but nonspecific, the azidoTag/click methodology had low sensitivity, and the dansylTag methodology failed to detect modified proteins in hepatocytes. The COATag methodology was judged superior, detecting 5 ng of modified protein in vitro and identifying protein targets in hepatocytes. In metabolism studies in rat liver microsomes, the azide group was metabolically labile, which was a contributing factor in not selecting the azidoTag methodology in the oxidative environments required for bioactivation. For study of the protein targets of electrophilic metabolites formed by in situ oxidative bioactivation, the COATag is both sensitive and specific and does not appear to suffer from poor cell permeability.