Expanding the Scope of a Chemical Modification and Mass Spectrometry-Based Method For Protein-Ligand Binding Analysis

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With support form the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry, Professor Michael Fitzgerald and his research group at Duke University will develop and apply new quantitative mass spectrometry-based proteomics methods for the large-scale thermodynamic analysis of protein-ligand binding interactions. The specific protocols under development utilize the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with a stable isotope labeling with amino acids in cell culture (SILAC) approach to compare the equilibrium unfolding/folding properties of proteins and protein ligand-complexes in real biological mixtures (e.g., cell lysates). The new protocols, which are designed to significantly expand the number of proteins assayed in proteome-wide SPROX experiments, will be benchmarked in several proof-of-principle experiments in which the endogenous proteins in a yeast cell lysate are analyzed for binding to ATP (a ubiquitous enzyme cofactor), to geldanamycin (an Hsp90 inhibitor), and to cyclosporine A (an immunosuppressive agent that directly binds cyclophilin A and indirectly interacts with calcineurin. The new protocols will also be used in a real drug mode-of-action studies involving manassantin A, which is a natural product with demonstrated anti-cancer activity in cell-based assays but an as yet unknown mode-of-action.The strategies and protocols to be created will be readily transferable to almost any protein-ligand system or biological mixture regardless of the ligand (i.e. small molecule, DNA, peptide, or other proteins) or binding affinity. The research provides interdisciplinary research experiences for both graduate and undergraduate students, and as well as high school students from the North Carolina Project SEED program, which is an on-going science enrichment program designed to steer disadvantaged, underrepresented minority students from North Carolina high schools into chemistry and chemistry-related careers.