Application of proteomics mass spectrometry to the Keap1/Nrf2 chemoprevention pathway

Up-regulation of cytoprotective enzymes is an important means of protecting cells against reactive oxygen species and electrophilic xenobiotics that can initiate and promote carcinogenesis. The induction of these enzymes can be mediated by the Keap1-Nrf2 oxidative stress system. In this dissertation, proteomic mass spectrometry was used to help clarify the mechanisms of the Keap1-Nrf2 oxidative stress system.;The first part of this dissertation concerned the investigation of cysteine modification in human Keap1 by electrophilic compounds. An LC-MS/MS method was developed to characterize the reactivity of human Keap1 towards natural products that up-regulate the ARE including xanthohumol, isoliquiritigenin and 10-shogaol. Although the Keap1 alkylation pattern was different for each electrophile, C151 was always detected among the top three most reactive cysteines by all three chemoprevention agents. These in vitro results indicate that C151 is the most important site of alkylation on Keap1 by chemoprevention agents that function by activating the ARE through Nrf2. Moreover, an immobilized Keap1 antibody assay was developed based on magnetic beads and nano LC-MS/MS to study the Keap1 modification in cell samples. This assay showed very good selectivity in complex matrices and could facilitate the study of how alkylation of Keap1 induces chemoprevention agents in cells.;In addition to the identification of Keap1 modification, the phosphorylation of Nrf2 was studied. Ser-40 was identified as phosphorylated after incubation with PKC in vitro using both MALDI and electrospray mass spectrometry. An LC-MS based method was also developed to quantify the phosphorylation at Ser-40. Next, a relative protein quantification method was developed based on stable-labeling. The method was validated using standard protein mixtures and showed good sensitivity and accuracy for both protein identification and quantitation.;Taken together, these proteomics mass spectrometry studies have provided insight into the mechanism of the Keap1-Nrf2 signal pathway. In addition, all of the new proteomics methods developed during this dissertation will be applied to cell culture experiments in future studies. These studies will provide more evidence to elucidate the mechanism of action of chemopreventive agents that up-regulate the cytoprotective enzyme expression by ARE and should provide guidance for developing effective agents to prevent cancer.