Native Mass Spectrometry of Protein Complexes

Before MS analysis, soluble proteins were buffer-exchanged into 200 mM ammonium acetate pH 8.0, and membrane proteins were buffer-exchanged into 200 mM ammonium acetate at various pH values, with twice the CMC (critical micelle concentration) of the detergent of interest. Data were collected on a modified QExactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) optimized for analysis of high-mass complexes, using methods previously described for membrane proteins (25 (link)). Data were analyzed using Xcalibur 3.0 (Thermo Fisher Scientific) and UniDec software (51 (link)). The relative intensities of monomers and dimers were obtained by deconvoluting the native MS data using UniDec and were converted to mole fractions to determine the monomer and dimer concentrations at equilibrium. To obtain the monomer-dimer equilibrium constants, a previously established monomer-dimer model was used (33 (link)). Detailed descriptions of the methodology for protein expression constructs, protein expression and purification, native mass spectrometry, and isothermal titration calorimetry are described in SI Appendix, Materials and Methods.