Hydrogen deuterium exchange followed by Mass Spectrometry (HXMS) is a biophysical tool capable of probing protein/ligand interactions, conformational changes, and protein dynamics. Despite an increased number of applications, the expansion of the technology has been slowed by its intrinsic technical and analytical complexity (i.e., sequential digestion at pH 2.5 and rapid HPLC separation at 0°C). Although many HXMS studies have been conducted with electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) mass spectrometry turns out to be a convenient tool for this purpose. MALDI-HXMS combines the advantages of high speed of analysis and excellent sensitivity and accuracy of mass measurements with the capability to analyse peptide maps in a single spectral acquisition. With recent advancements in sample preparation robotics entire MALDI-HXMS experiments (typically 64-128 samples) can be executed in less than one hour.
We have developed an integrated R-package named MALDI-HDX to help analyse large MALDI-HXMS datasets. MALDI-HDX uses the MALDIquant Foreign R package to import Bruker MALDI-TOF data. A viewer facilitates semi-automated assessment of the quality of all spectral segments across an XIC peak for centroid mass determination. The resulting HXMS data are then subjected to statistical analysis using the MEM-HDX R package. This interactive tool allows the user to validate, visualize and compare the relative deuterium incorporation on the amino acid sequence and 3D structure, providing both spatial and temporal information.
The biophysical characterization of therapeutic monoclonal antibodies recognizing different epitopes establishes the ability of MALDI-HXMS to provide useful information. This approach may be extended to supramolecular interactions, provided that peptide mapping is conducted carefully in order to deliver unambiguous sequence coverage. MALDI-MS alone might not be accurate enough to achieve successfully this task in all cases, and should then be associated to LC-MS/MS sequence assignment for the first part of HXMS methodology.