The Calcium-Modulated Structures of Calmodulin and S100b Proteins are Useful to Monitor Hydrogen/Deuterium Exchange Efficiency Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Abstract

Hydrogen/deuterium exchange (HDX) using matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry is a sensitive, salt-tolerant and high-throughput method useful to probe protein conformation and molecular interactions. However, a drawback of the MALDI HDX technique is that sample preparation methods can typically result in higher levels of artificial deuterium in-exchange and/or hydrogen back-exchange just prior to or during mass analysis; this may impair data reproducibility and impede structural and kinetic data interpretation. While methods to minimize effects of back-exchange during protein analyte deposition on MALDI plates have been reported, this study presents a readily available, highly sensitive protein control set to facilitate rapid MALDI HDX protocol workup. The Ca2+-induced solvent accessible surface area (ASA) changes of calmodulin (CaM) and S100 proteins were employed to monitor and optimize HDX protocol efficiency. Under non-stringent room temperature conditions, the Ca2+-induced deuterium exchange of CaM, ΔDca2+, MH+ shifts −17 Da to −24 Da, while S100 ΔDca2+ MH+ shifts +8 Da to +12 Da. By comparing the divergent CaM and S100 Ca2+-induced deuterium mass shift differences, HDX sample workup and MALDI plate spotting conditions can easily be monitored.