SILAC-Based Strategy for Proteome-Wide Analysis of Protein-Ligand Binding Interactions (Part 2/2)

A quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method, which is general with respect to ligand, measures the ligand-induced changes in protein stability associated with protein-ligand binding. The methodology is demonstrated in a proof-of-principle study in which the well-characterized protein-drug interaction between cyclosporine A and cyclophilin A is analyzed in the context of a yeast cell lysate. The method is also utilized for the global analysis of adenosine triphosphate (ATP) binding to proteins in the yeast proteome using the non-hydrolyzable ATP analogue, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate.