Development of Software for the In-Depth Analysis of Protein Dynamicsas Determined by MALDI Mass Spectrometry-Based Hydrogen/DeuteriumExchange

摘要

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful forrapidly analyzing the kinetic properties of small amounts of protein. However,the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) massspectrometry (MS) is time-consuming due to a lack of dedicated software.Currently available software programs mainly calculate average mass shifts, eventhough the isotopic distribution width contains information regarding multipleprotein conformations. Moreover, HDX reaction samples are typically composed ofpeptides that contain various numbers of deuterium atoms, which also hinders therapid and comprehensive analysis of protein dynamics. We report here on thedevelopment of a software program “Scipas DX” that can be used to automaticallyanalyze the hydrogen–deuterium isotopic distribution in peaks in HDX spectra andcalculate the average number of atoms exchanged, the average deuteration ratio,the abundance ratio for exchanged atoms, and their fitted spectra with a highdegree of accuracy within a few minutes. Analysis of the abundance ratio forexchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicatethat the local structure at residues 83–106 and 107–117 are in a slowequilibrium, suggesting that these regions adopt multiple conformations that areinvolved in the stability and in switching between the active and inactiveforms. Furthermore, precise HDX kinetics of the average deuteration ratio bothconfirmed the known induced conformations of two regions (residues 46–75 and131–165) that are responsible for ligand binding and verified the novelstructural dynamics of residues 107–117 and 166–196 following ligand binding toligand-binding pockets 1 and 2, respectively. Collectively, these resultshighlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-basedanalyses of protein dynamics.