Response Factor Correction for Quantitative Determination of Homooligomeric Protein Binding to DNA by Native Protein Mass Spectrometry

Over the past two decades, native protein mass spectrometry (nMS) has emerged as a complementary approach for elucidating molecular parameters of biological complexes including stoichiometry and thermodynamic binding. Being able to simultaneously quantify distinct components within heterogeneous mixtures, while also requiring small amounts of material and minimal sample preparation provide numerous advantages over traditional solution-phase biochemical techniques. However, several caveats related to species specific MS signal responses, including changes in response factors (RFs) upon biological complex formation, have not been fully resolved, which has prevented the technique from being widely adopted. Herein, we utilize nMS to determine the stepwise binding affinities of a recombinant single-strand DNA binding protein (SSB) from Saccharolobus solfataricus (Sso) along single-stranded DNA (ssDNA). This work resolves conflicting previous biochemical reports on the stoichiometry and affinities of SsoSSB, while also highlighting the advantages and limitations of nMS quantification. Covalent concatemers of SsoSSB were used to compare RFs of each oligomer as a function of molecular weight and to correct for physical and instrumental parameters that systematically underrepresent abundances of larger mass species. Furthermore, we show that regardless of quantification metric (peak area or intensity) utilized for the nMS data, meaningful biochemical data can be extracted from multicomponent biochemical systems. Importantly, binding affinities determined by nMS are in close agreement with measured bulk solution-phase measurements but have the added benefit of separately quantifying individual binding steps within a multistep assembly process to determine thermodynamic properties of cooperativity.