Analysis of the Ubiquitin Microenvironment via Photocatalytic Dexter Energy Transfer

• Phillip Moquin, Biochemistry, University of Delaware

• Zhihao Zhuang, Chemistry and biochemistry, University of Delaware

Previously, the synthesis of an iridium photocatalyst was reported for mapping the localized microenvironment of antibodies of interest, including the identification of antibody binding targets and other associated biomolecules. The novel micromapping method involved the synthesis of a biotinylated diazirine as well as the aforementioned iridium photocatalyst, which, upon conjugation to an antibody, could, by means of a Dexter energy transfer upon photoirradiation, produce localized carbene generation, enabling labeling (biotinylation) of biomolecules in the diffusion range of the short-lived carbene species. The utilization of carbenes allows for an especially high precision localization of micromapping, owing to their severe instability, especially in aqueous systems where immediate quenching by water greatly limits the carbene diffusion radius. In this work, I report the successful synthesis in progress of a similar iridium photocatalyst analog, as well as the reported diazirine for use in the microenvironment mapping of various ubiquitin species. After the completed synthesis, labeling, streptavidin pull-down, proteolytic digestion, and subsequent proteomic analysis via LC-MS will serve to reveal the scope of biomolecules potentially associated with ubiquitination pathways. Applying this high-resolution method of micromapping to ubiquitin species will serve to develop a further understanding of ubiquitin pathways, potentially giving way to new leads in designing more targeted approaches to ubiquitin-related investigation, especially in the realm of therapeutics.