Hydrogen-Deuterium Exchange Analyzed by Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry and the HET-s Prion Model

Abstract

Hydrogen/deuterium (H/D) exchange analyzed by mass spectrometry (HXMS) is a valuable tool for the investigation of protein conformation and dynamics. After exchange, the sample is generally submitted to electrospray ionization for mass analysis. Matrix‐assisted laser desorption ionization (MALDI) has been used in a limited number of studies but has several significant advantages that include simplification of the spectra attributable to a predominance of singly charged ions, speed of analysis, sensitivity, and low H/D back‐exchange level. MALDI‐HXMS has been used to study amyloid aggregates from the HET‐s prion protein. Our results underline the ability of this method to determine solvent accessibility within the amyloid aggregates, reaching a resolution of one to four amino acids. To achieve a complete peptide mass fingerprint of the protein, we have taken benefits of an ion trap operating in liquid chromatography‐MS/MS mode. MALDI time‐of‐flight‐MS was then used to determine deuterium incorporation within each peptide along the sequence of HET‐s. The combined advantages of these two instruments yield a suitable solution for HXMS experiments that require highly resolved peptide mass fingerprints, high sensitivity, and speed of analysis for deuterium incorporation measurements.