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 deuteriumlabeled matrix, which allows correct determination of the number ofpossible 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-MSimaging. 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.

Automated summary

This study developed a highly efficient hydrogen-deuterium exchange technique for more accurate metabolite identification in MALDI-MS imaging, while retaining spatial information. By combining deuterium-labeled matrix and D2O vapor, an HDX efficiency of 73-85% was achieved. This method can systematically improve untargeted metabolite annotation through METASPACE annotation.