Quinone methide (QM) is a short-lived reactive intermediate thought to have both cytotoxic and cytoprotective properties. The chemopreventive aspects of QM-releasing NO-aspirin have been previously highlighted and it is important to recognize the therapeutically relevant effects of QM on cellular mechanisms and pathways. This study investigates modification of important cellular proteins by QM to better understand the pathways linked with cytotoxicity and cytoprotection. Upon esterase bioactivation, NO-aspirins such as pNO-ASA and pBr-ASA release QM. Covalent modification of glutathione S-transferase P1 (GST P1) and Keap1 by QM was analyzed qualitatively and quantitatively with LC-MS/MS. Nuclear translocation of Nrf2, a consequence of Keap1 modification, was also studied with confocal fluorescence microscopy after treatment of cells with pNO-ASA. Colon cancer cells were treated with azido-pNO-ASA to identify and map the QM-modified proteins. Modified proteins were clicked to biotin to enable their separation from the unmodified proteins using streptavidin beads. The LC-MS/MS analysis showed over 150 QM-modified proteins from HT-29 cells including ALDH1, ENO1, HSP60, HSP90, PKM2 etc. with over 25% sequence coverage. In addition, QM modification of NF-?B (p65) and �-catenin was confirmed with western blotting while the QM modification of IKK was also investigated to understand pathways contributing to anti-inflammatory and chemopreventive activity of the parent drug.

Analysis of quinone methide protein targets with biotin pull-down and LC-MS/MS

244th National Meeting (2012)

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