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

Protein Sci. 2006 Jun;15(6):1465-75. doi: 10.1110/ps.062083406.

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 microM. Differences in DeltaDeltaG of dissociation between variants were determined with precisions < 0.1 kcal/mol. The DeltaDeltaG 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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Ions
  • Oxidation-Reduction
  • Protein Interaction Mapping / methods*
  • Ribonucleases / chemistry
  • Ribonucleases / metabolism
  • Solutions
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Urea / chemistry

Substances

  • Bacterial Proteins
  • Ions
  • Solutions
  • barstar protein, Bacillus amyloliquefaciens
  • Urea
  • Ribonucleases
  • Bacillus amyloliquefaciens ribonuclease