This project proposes the development of a new analytical system capable of performing single-cell assays of E3 ligase activity, a critical element in the ubiquitin proteasome system. This will be accomplished through the development of a novel peptide-based fluorescent E3 ligase reporter and technical improvements to an existing chemical cytometry platform. This E3 ligase reporter incorporates a degradation signal, or degron, which is recognized and ubiquitinated by an E3 ligase, as well as a fluorescent tag to facilitate detection. The reporter will be analyzed using a chemical cytometry platform employing capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The motivation for the development of an E3 ligase reporter is to generate a new tool for investigating E3 ligase activity in clinical tumor samples, with a focus on acute myeloid leukemia. Initial development efforts in the project will focus on methods development and optimization of the electrophoretic separation and detection of ubiquitinated and non-ubiquitinated forms of the reporter. Following the establishment of optimized separation conditions, the reporter will be validated and characterized in cell lysates generated from cell lines derived from patients with AML and other cancers known to exhibit dysregulated E3 ligase activity. Once this technology has been validated in cell lysates, the reporter will be modified to incorporate a myristoyl group, allowing for passive loading of the reporter into single cells. While cells lines will be employed for this technology development grant, the ultimate goal will be to use this technology to detect dysregulation of the UPS in primary clinical samples.