Fig. 2.

Establishing methodology for measuring the effects of a ligand on the target protein MDM2 using hydrogen-deuterium exchange mass spectrometry. MDM2 protein (2 μm final concentration) was assembled with DMSO or with Nutlin-3 (8 μm) at room temperature for 30 min, as described previously for the N-terminal domain of MDM2 (amino acids 1–126) (32). The reactions were then diluted with D₂O by sequentially adding, slowly with mixing, 5.14 μl, 10 μl, 20 μl, and 27 μl of D₂O. The reactions were incubated from 40 to 18,000 s and quenched with 3 μl of 0.87 m HCl with 1 m Glycine, frozen, and processed for pepsinization as in the materials and methods. The deuterium exchange rates of individual peptides (supplemental Fig. S1) is summarized using the HDX exchange plots for the 300-s deuteration time course.