Development of Software for Deep Analysis of Protein Dynamics as Determined by MALDI Mass Spectrometry-Based Hydrogen/Deuterium Exchange

Abstract

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetics of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to the lack of dedicated software. Further, existing software programs mainly calculate the average mass shift, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, the HDX reaction sample is composed of peptides incorporating various numbers of deuterium atoms, which also hinders rapid and comprehensive analysis of protein dynamics. We developed the software program “Scipas DX” to automatically analyze the hydrogen-deuterium isotopic distribution of peaks in HDX spectra and calculate the average number of atoms exchanged, average deuteration ratio, abundance ratio for exchanged atoms, and their fitted spectra with high accuracy in a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX detected slow equilibrium of the local structure at residues 83-106 and 107-117, revealing that these regions adopt multiple conformations involved in the stability and in the functional switch between the active and inactive form. Furthermore, precise HDX kinetics of the average deuteration ratio both confirmed the known induced conformations of two regions (residues 46-75 and 131-165) responsible for ligand binding and verified the novel structural dynamics of residues 107-117 and 166-196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analysis of protein dynamics.