Paper TM+AS-MoM4
Spatially-resolved Tandem Mass Spectrometry Increases Molecular Confidence in a Multimodal Mass Spectrometry Imaging Investigation of a Tripartite Plant-fungus-cyanobacteria Interaction

Monday, October 30, 2017, 9:20 am, Room 5 & 6

Plant microbiomes represent a complex mix of interacting species with diverse physiologies and phylogenetic origins. Their functional outcomes are critical to biogeochemical cycles, yet measuring molecular (e.g., metabolite) exchange among interacting species is a major technical challenge. Traditional bulk metabolomic technologies are often limited in their ability to distinguish between molecules that remain localized within microbes and exuded molecules that are in proximity, thus often disregarding the multifaceted chemical exchange within and between interacting species. Mass spectrometry imaging (MSI) methodologies have been recently adopted to visualize the flow of metabolites produced by agar-supported microbial colonies. Several ionization modalities are suitable for MSI of microbial communities, with matrix-assisted laser desorption/ionization (MALDI) being most commonly used. When coupled with ultra-high resolution mass analyzers (e.g., Fourier transform ion cyclotron resonance mass spectrometers; FTICR-MS), these imaging sources offer the high mass resolution and accuracy needed for putative identification of metabolites in individual pixels in the image. However, orthogonal methodologies (e.g., tandem MS) are often required for confident metabolite identification.

Herein, we explored the interactions within a tripartite system of moss, cyanobacteria, and fungus using a multimodal imaging strategy, which employs liquid extraction surface analysis (LESA) tandem MSI to examine previously MALDI imaged samples. This method improved exometabolite identification confidence by preserving spatial dimensionality in the tandem MS experiment. Specifically, we found the combination of these two imaging modalities generated very congruent mass spectral information, providing the link between highly accurate structural information offered by LESA and high spatial resolution attainable by MALDI. Finally, FTICR-based secondary ion mass spectrometry provided new insights into tripartite community using correlative fragment data (SIMS and LESA-MS/MS), while delivering higher lateral resolution MS images. These multimodal imaging results offer detail metabolic insights into a moss, cyanobacterium, and fungus in isolation and when in a tripartite symbiosis.