New mass spectrometry tools for characterizing protein-ligand interactions

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The objectives of this proposal are the development and application of new mass spectrometry (MS) methods to detect and characterize protein-ligand interactions. The proposed research will focus on three areas: (i) Discovery and characterization of host-cell receptors. The interactions of pathogen-generated proteins with carbohydrate receptors on the surfaces of epithelial cells are critical events in many infectious diseases. However, the identification of the functional receptors remains a significant challenge. The goal of the planned research is to develop a novel catch-and-release electrospray ionization (ESI) MS assay for detecting specific interactions between pathogen-generated proteins and their carbohydrate receptors in cell membranes. Notably, the assay will employ direct ESI-MS analysis of aqueous solutions of receptor-binding proteins and receptors, which are incorporated into nanodiscs (NDs). Application of this assay to NDs prepared from lipids extracted from cell membranes will allow for the discovery of human receptors of a variety of bacterial and viral proteins. (ii) Quantifying ligand binding to large protein assemblies. The direct ESI-MS binding assay is an established tool for quantifying protein-ligand binding in solution. However, the requirement that the free and ligand-bound protein ions can be directly detected and accurately quantified imposes limitations on the interactions that can be investigated. The goal of this project is to develop new ESI-MS assays to quantify ligand interactions with large protein complexes, such as virus particles, in vitro. (iii) Elucidating the molecular origin of hydrophobic protein-ligand interactions. Hydrophobic bonding plays important roles in many biochemical processes, such as protein folding and non-covalent association. However, a quantitative description of the underlying forces is lacking. The goal of the proposed research is to develop a more complete description of the interplay between intrinsic interactions and solvent effects in hydrophobic protein-ligand interactions.