Summary/Abstract The reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are key signaling molecules that mediate diverse biological processes, including cell migration involved in tissue repair, immune response, and cancers. The central molecular targets of ROS are protein cysteine residues that form various thiol oxoforms, including S-glutathionylated cysteines, termed as S-glutathionylation. This protein S-glutathionylation regulates protein activity in a number of signaling pathways. Despite the continuing advance on identification of glutathionylated proteins, identification of the specific glutathionylated cysteines that control definite biological functions has been challenging. To provide the insights into the glutathionylation-susceptibility of global cysteines, we have developed a chemical proteomic approach, termed clickable glutathione, that enables to study S- glutathionylation. In this proposal, we will develop an integrative strategy combining our chemical proteomic platforms with functional biological analyses to streamline identification of glutathionylation-susceptible cysteines that control cell migration. First, we aim to identify glutathionylation-sensitive cysteines in mammalian cell lines during cell migration induced by D-amino acid oxidase (DAAO) with D-Ala, which produces spatiotemporal and magnitude-controlled levels of H2O2. We will use our quantitative proteomics and bioinformatic analyses to identify a group of cysteines highly susceptible to glutathionylation and functionally related to migration. Because of the importance of localized H2O2 production, the strategy will be extended to the use of localized DAAO/D-Ala systems to determine localization-dependent glutathionylation of global cysteines. Second, we aim to determine regulatory roles of the identified glutathionylated cysteines in cell migration. In preliminary studies, we identified the redox-active glutathionylated cysteines in three proteins, PP2Cα, ARHGEF7, and NISCH, which increase cell migration in functional analyses. We will investigate glutathionylation-susceptibility of three proteins and their downstream signaling pathways mediated by glutathionylation. Lastly, we will apply a combination of chemical proteomics, bioinformatics, and functional screening analyses to find new glutathionylation-susceptible proteins that regulate cell migration.