Hydrogen-Deuterium Exchange Mass Spectrometry of 14-3-3ζ

HXMS experiment was performed as described previously 53 (link). Briefly, deuterium labeling was initiated with a 20-fold dilution into D₂O buffer of a pre-equilibrated (30 min, RT) aliquot of 14-3-3ζ protein and 14-3-3ζ protein with PTA stock solution. The labeling reaction was quenched with the addition of quenching buffer (37.5% [v/v] hydrochloric acid) at indicated times (0.25, 1, 10, 20, 60 and 240 min). Subsequently, samples were injected and online digested by a Waters ENZYMATE BEH pepsin column (2.1×30 mm, 5µm). The peptides were then trapped and desalted on a VanGuard Pre-Column trap (ACQUITY UPLC BEH C18, 1.7 µm) for 3 min, eluted in the trap using 15% acetonitrile at a flow rate of 100 µL/min, and separated using an ACQUITY UPLC BEH C18 column (1.7µm, 1.0×100 mm). Relative deuterium levels of all peptides were calculated by subtracting the mass of the undeuterated control sample from that of the deuterium-labeled sample. All mass spectra were analyzed using DynamX 3.0. Deuterium levels were not corrected for back exchange and thus reported as relative.