Improving hydrogen/deuterium exchange mass spectrometry by reduction of the back‐exchange effect

The measurement of deuterium incorporation kinetics using hydrogen/deuterium (H/D) exchange experiments is a valuable tool for the investigation of the conformational dynamics of biomolecules in solution. Experiments consist of two parts when using H/D exchange mass spectrometry to analyse the deuterium incorporation. After deuterium incorporation at high D 2 O concentration, it is necessary to decrease the D 2 O concentration before the mass analysis to avoid deuterium incorporation under artificial conditions of mass spectrometric preparation and measurement. A low D 2 O concentration, however, leads to back‐exchange of incorporated deuterons during mass analysis. This back‐exchange is one of the major problems in H/D exchange mass spectrometry and must be reduced as much as possible. In the past, techniques using electrospray ionization (ESI) had the lowest back‐exchange values possible in H/D exchange mass spectrometry. Methods for the measurement of H/D exchange by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) that have been developed since 1998 have some significant advantages, but they could not achieve the back‐exchange minima of ESI methods. Here, we present a protocol for H/D exchange MALDI‐MS which allows for greater minimization of back‐exchange compared with H/D exchange ESI‐MS under similar conditions. Copyright © 2003 John Wiley & Sons, Ltd.