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

¹ Program in Biophysics
² Department of Chemistry
³ Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, California 94720-1460, USA

(RECEIVED January 6, 2006; FINAL REVISION March 3, 2006; ACCEPTED March 13, 2006)

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 G of dissociation between variants were determined with precisions <0.1 kcal/mol. The G 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.

Keywords: mass spectrometry; protein–protein interaction; barnase; electrospray; biosensing

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