Deuterium Exchange Kinetics of PDK1

The experiments were performed essentially as previously described for studies on the catalytic domain of PDK1 (84 (link)). In short, His-PDK1_1-556 and His-PDK1_50-359 were diluted in buffer containing deuterium oxide ([²H]₂O) at 30°C for the indicated times. The exchange was stopped by quenching with acid ((0.5% TFA, 5 M GnCl) to achieve a final pH of 2.5) and cooling to 0°C. Each sample was immediately injected into nanoUPLC HDX Sample Manager (Waters) for online pepsin digestion using Poroszyme pre-packed pepsin column (Thermo Fisher Scientific), using 0.1% formic acid in LC-MS water at 100 μl/min, then trapped and desalted using a VanGuard C-18 column (Waters), followed by reverse-phase separation using ACQUITY™ 2.1 X 5 mm BEH C-18 column (Waters) using a 0.1% formic acid in acetonitrile gradient. The labelled derivative peptides were analyzed by Synapt G2 Si mass spectrometer (Waters) operating in positive ion mode using an MSE acquisition method. The mass spectrometer was continuously calibrated using 200 fmol/μl Glu-fibrinopeptide B standard flowing at 1 μl/min. The graphics represent the number of deuterium atoms incorporated into each peptide during the incubation.