Thermodynamic Analysis of Protein Stability and Ligand Binding Using a Chemical Modification- and Mass Spectrometry-Based Strategy

Described here is a new technique, termed SPROX (stability of proteins from rates of oxidation), that can be used to measure the thermodynamic stability of proteins and protein−ligand complexes. SPROX utilizes hydrogen peroxide in the presence of increasing concentrations of a chemical denaturant to oxidize proteins. The extent of oxidation at a given oxidation time is determined as a function of the denaturant concentration using either electrospray or matrix-assisted laser desorption/ionization mass spectrometry. Ultimately, the denaturant concentration dependence of the oxidation reaction rate is used to evaluate a folding free energy (ΔG_f) and m value (δΔG_f/δ[Den]) for the protein’s folding/unfolding reaction. Measurements of such SPROX-derived ΔG_f and m values on proteins in the presence and absence of ligands can also be used to evaluate protein−ligand affinities (e.g., ΔΔG_f and K_d values). Presented here are SPROX results obtained on four model protein systems including ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). SPROX-derived ΔG_f and m values on these proteins are compared to values obtained using more established techniques (e.g., CD spectroscopy and SUPREX). The dissociation constants of several known protein−ligand complexes involving these proteins were also determined using SPROX and compared to previously reported values. The complexes included the CypA−cyclosporin A complex and the BCAII−4-carboxybenzenesulfonamide complex. The accuracy and precision of SPROX-derived thermodynamic parameters for the model proteins and protein−ligand complexes in this study are discussed as well as the caveats of the technique.