A Practical Workflow for Reliable MALDI-Based Hydrogen/Deuterium Exchange Mass Spectrometry

Matrix-assisted laser desorption/ionization-based hydrogen/deuterium exchange mass spectrometry (MALDI-HDX-MS) can provide peptide-level information on protein conformational dynamics without any specialized liquid chromatography (LC)-HDX instrumentation. Accurate interpretation of structure-dependent deuterium incorporation and reproducible MALDI-HDX measurements require minimization of unintended exchange and spectral variability during MALDI sample preparation and acquisition. Appropriate control of matrix crystallization, humidity, and spectral acquisition timing is therefore essential for reliable MALDI-HDX analysis. In this Technical Report, we describe a practical workflow for reliable MALDI-HDX measurements using human adenylate kinase 1 (AK1) as a model protein. The workflow includes humidity-controlled spotting on a precooled MALDI plate, preparation of a precooled α-cyano-4-hydroxycinnamic acid matrix solution, rapid vacuum-assisted drying, and time-controlled spectral acquisition. Precooling of the matrix solution induced visible two-layer separation, and use of the lower layer produced relatively uniform, small crystal clusters and favorable MALDI spectra. The influence of acquisition timing after matrix drying was evaluated using AK1 peptic fragments. For a peptide fragment sensitive to post-drying forward exchange, a small but detectable increase in deuterium incorporation was observed at 15 min after the start of matrix drying, whereas no substantial change was observed at 10 min compared with 5 min. Importantly, the increase caused by delayed acquisition was smaller than the difference in deuterium incorporation between structurally distinct peptide regions, indicating that structure-dependent HDX information was retained within the defined acquisition window. Establishment of practical matrix crystallization and spectral acquisition conditions will facilitate the reliable application of MALDI-HDX-MS to biological studies of protein conformational dynamics.