Free energies of protein–protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods

Abstract

The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solution‐phase affinities of tightly bound protein–protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form protein–protein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution‐phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations <25 μM. Differences in ΔΔG of dissociation between variants were determined with precisions <0.1 kcal/mol. The ΔΔG values obtained deviate on average by 0.26 kcal/mol from those measured with a solution‐phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein–protein‐binding surfaces for complexes that have affinities in the picomolar to nanomolar range.