Prior to analysis, the protein was buffer exchanged into 0.2 M ammonium acetate (pH 6.8) and diluted to 10 μM. DTT was dissolved in water and prepared at a 400 mM stock. Each ligand was dissolved in ethanol and diluted to 10× stock concentrations. The final mixture was prepared by adding 4 μL protein, 0.5 μL DTT stock, and 0.5 μL ligand stock for final concentration of 4 mM DTT and 8 μM protein. Final ligand concentrations were used as annotated. The mixtures were then incubated for 10 min at room temperature prior to analysis. Each sample was mixed and analyzed in triplicate.
Native MS was performed using a Q-Exactive HF quadrupole-Orbitrap mass spectrometer with the Ultra-High Mass Range research modifications (Thermo Fisher Scientific). Samples were ionized using nano-electrospray ionization in positive ion mode using 1.0 kV capillary voltage at a 150 °C capillary temperature. The samples were all analyzed with a 1000–25,000 m/z range, the resolution set to 30,000, and a trapping gas pressure set to 3. Source fragmentation (10–50 V) was applied to all samples to aid in desolvation. Data were deconvolved and analyzed with UniDec.41 (link)
Native MS was performed using a Q-Exactive HF quadrupole-Orbitrap mass spectrometer with the Ultra-High Mass Range research modifications (Thermo Fisher Scientific). Samples were ionized using nano-electrospray ionization in positive ion mode using 1.0 kV capillary voltage at a 150 °C capillary temperature. The samples were all analyzed with a 1000–25,000 m/z range, the resolution set to 30,000, and a trapping gas pressure set to 3. Source fragmentation (10–50 V) was applied to all samples to aid in desolvation. Data were deconvolved and analyzed with UniDec.41 (link)
