Efficient Hydrogen–Deuterium Exchange in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging for Confident Metabolite Identification

Highly efficient hydrogen–deuterium exchange (HDX) is developed for mass spectrometry imaging (MSI) with low-vacuum matrix-assisted laser desorption/ionization (MALDI). A HDX efficiency of 73–85% is achieved by introducing D2O vapor into a heated MALDI source in combination with a deuterium-labeled matrix, which allows correct determination of the number of possible H/D exchanges for up to 17 labile hydrogens. This provides valuable orthogonal information to supplement m/z, allowing for increased confidence in metabolite identification while retaining the spatial information MSI supplies. When combined with high-throughput METASPACE annotation, this approach can systematically improve untargeted metabolite annotations in MALDI-MS imaging. The developed method was applied to MALDI-MS imaging of the top surface, bottom surface, and middle section of Lemna minor fronds. Out of a total of 56 on-sample annotations made with the BraChem database using a 10% false discovery rate, 31 of these annotations (55%) matched our HDX data, providing additional confidence. For the remaining 45%, our data allowed us to narrow down structural possibilities and eliminate incorrect structures, greatly increasing confidence in metabolite identification.