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
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
