Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of structurally identified and yet-undefined metabolites across tissue cryosections. While numerous software packages enable pixel-by-pixel imaging of individual metabolites, the research community lacks a discovery tool that images all metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs informs discovery of unanticipated molecules contributing to shared metabolic pathways, uncovers hidden metabolic heterogeneity across cells and tissue subregions, and indicates single-timepoint flux through pathways of interest. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling and instrument drift, markedly enhances spatial image resolution, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent tool to enhance knowledge obtained from current spatial metabolite profiling technologies.