Ultra high mass range research modifications

Before MS analysis, the protein
was buffer exchanged using two Micro Bio-Spin columns (Bio-Rad) and
diluted into 0.2 M ammonium acetate to a concentration of 10 μM.
Each drug was diluted with 100% ethanol to concentrations of 100,
200, and 400 μM. Imidazole, a charge reducing reagent, was added
to each sample to stabilize the drug-bound state at a final concentration
of 40 mM. For each sample, 0.5 μL of ligand was added and dried
down in each tube prior to the addition of 0.5 μL of 40 mM DTT,
0.5 μL of 400 mM imidazole, and 4 μL of protein for a
final concentration of 4 mM DTT and 8 μM protein. Final ligand
concentrations were either 10, 20, or 40 μM.
Native mass
spectrometry (MS) was performed using a Q-Exactive HF quadrupole-Orbitrap
mass spectrometer with the Ultra-High Mass Range research modifications
(Thermo Fisher Scientific) as described in our previous publications.^{7 (link),8 (link)} Data were deconvolved and analyzed with UniDec.²¹

Before 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 of protein, 0.5 μl of DTT stock, and 0.5 μl of ligand stock for a final concentration of 4 mM DTT and 8 μM protein. Final ligand concentrations were 10 μM. The mixtures were then incubated for 10 min at room temperature before analysis. Each sample was mixed and analyzed in triplicate.
Native mass spectrometry 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 to 25,000 mass/charge ratio (m/z) range, the resolution set to 30,000, and a trapping gas pressure set to 3. Between 10 and 50 V of source fragmentation was applied to all samples to aid in desolvation. Data were deconvolved and analyzed with UniDec (42 (link)).

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 10X 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 10 μM. The mixtures were then incubated for 10 minutes at room temperature prior to analysis. Each sample was mixed and analyzed in triplicate.
Native mass spectrometry (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 1,000–25,000 m/z range, the resolution set to 30,000, and a trapping gas pressure set to 3. Between 10 and 50 V of source fragmentation was applied to all samples to aid in desolvation. Data were deconvolved and analyzed with UniDec.^{43 (link)}