New Tools and Approaches for Understanding Metal-Mediated Interactions in the Extracellular Space

About This Grant

PROJECT DESCRIPTION Trace metal micronutrients, such as copper, iron, and zinc are vital to a host of biological processes, but their dyshomeostasis is associated with a number of disease states. The context in which a metal resides within a biological environment significantly influences its activity and function. Recent years have seen a rise in tools for monitoring metal ions and have illuminated the diversity in metal speciation in biology, but many of these tools are focused on probing metals in the intracellular space. The state-of-the-art methods for assessing metal status in extracellular fluids such as blood plasma focus either on absolute quantitation or evaluate a limited number of metal-containing species. While these methods have offered important insight into extreme cases of metal deficiency or overload, subtle imbalances are more challenging to diagnose and understand with the available methods. The objective of this research program is to expand and elucidate the metal speciation of the extracellular space, with an immediate focus on blood-based fluids due to their clinical importance in biomarker assessment. In previous work, we developed new bioluminescence-based probes and enrichment methods for mass spectrometry-based proteomics for identifying reactive copper pools and potential binding partners in serum. Additionally, we identified new interactions between transition metals and peptide hormones that influence their cellular behavior, quantitation in diagnostic assays, and interaction with other proteins in serum. In this proposal proposal, we seek to further develop the extracellular metal biochemical profile in the following ways. First, we seek to expand the toolbox for assessing extracellular metal interactions. Optical sensing approaches include the use of native albumin as a bioluminescence enzyme to pair with metal-responsive substrates and the development peptide-based detection platforms with metal-responsive cell surface interactions or internalization. Second, we will progress our metal-affinity enrichment strategies via on-resin digestion methods to gain new insight on metal chelate-interaction sites, with additional approaches that include incorporating pre-enrichment steps to identify metal-binding species in plasma subproteomes. Finally, we seek to expand the understanding of metal-mediated molecular interactions in blood plasma by developing and applying strategies to identify and investigate metal-mediated peptide/protein interactions these may influence hormone bioavailability and function. Objectives in this area include size-based fractionation methods develoepd and optimized with fluorescently-labeled peptide analogues, and catch-and-release peptide affinity strategies for identifying protein binding partners. The tools developed in this proposal will be applied to samples from collaborators, biorepositories, and mouse models to generate metal-centric biomarker profiles in metabolic disorders. The insights and technologies that emerge from this proposal will directly impact nutritional treatments, therapeutic peptide development, and biomarker discovery for metal status.