Abstract
Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied(1). Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples(1-5). This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.