Assessment of the Repeatability and Reproducibility of Hydrogen/deuterium Exchange Mass Spectrometry Measurements

A system to perform automated hydrogen/deuterium exchange mass spectrometry measurements was constructed using an XYZ robotic autosampler that was capable of performing solvent manipulations and a 4.7 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The system included features such as the first demonstration of a 'dual column' high-performance liquid chromatography (HPLC) setup, and a novel digestion strategy. The performance of the system, in terms of the repeatability and reproducibility of the measurement of protein hydrogen/deuterium exchange, was assessed over a 2-month period. The sensitivity of the measurement of hydrogen exchange towards several parameters was assessed, which allowed their impact on the reproducibility to be discussed. The parameters assessed were the temperature of the HPLC columns and switching valves, the temperature of the quench solutions, the pH of the mobile phase, the pH of the quenched solution, the acid used in the mobile phase and the analytical column used.