Representativenative mass spectra of CYP121 and selected compounds 26a, 25a, and 19a demonstrated thatcompounds formed stable complexes with both monomeric (m/z 5250–6750) and dimeric (m/z 5250–6750) CYP121. The number of boundligands (orange spheres) increased in a concentration-dependent mannerand was proportionate to the binding affinity (KD) of the compounds. Spectra were collected for CYP121 (8.7or 5 μM) at three different ligand-to-protein ratios (0.5:1,1:1, and 25:1) and in the presence of DMSO-d6 (2.2% or 5% v/v) alone. The concentration of protein or DMSO-d6 did not affect the quality of the spectraor ligand binding interactions, but higher concentrations of DMSO-d6 reduced the charge state of the protein. Ligandbinding stoichiometry was calculated from the difference in mass ofligand-bound and unbound (orange dashed lines) protein peaks dividedby the molecular weight of ligands (Table S1, Supporting Information).