DESI-MS imaging reveals spatial variations of secondary metabolites in Astragalus roots and their correlation with biosynthesis

Mass spectrometry imaging (MSI) has advanced the in situ analysis of chemical constituents in traditional Chinese medicine (TCM). However, many TCMs that are derived from perennial roots or rhizomes (e.g., ginseng, Astragalus, and licorice) present two main challenges: their hard texture hindering effective sectioning, and their secondary metabolites, particularly those with post-modification, are prone to unwanted molecular ion fragmentation, which reduces analytical sensitivity. In this study, Astragalus roots were used as a model to address these challenges in desorption electrospray ionization (DESI)-MSI. Firstly, poly-L-lysine moistening-cryosectioning method facilitated tissue sectioning and enhanced signal intensity, while optimizing the collision gas flow rate and scan time minimized undesired ion fragmentation. Applying this method to Astragalus roots revealed that isoflavones and their glycosides were widely distributed across all tissues, whereas saponins and organic acids exhibited distinct tissue-specific localization in the periderm and cambium, respectively. These spatial distributions were validated by LC/MS analysis. Integration with transcriptomic data revealed correlations between metabolite localization and the expression of their biosynthetic genes. Finally, application of the method to samples with different origins further revealed variations in the spatial distribution of isoflavones, including formononetin (17) and calycosin (12), as well as organic acids. Collectively, these findings provide an effective strategy for MSI analysis of TCMs and plant secondary metabolism, highlighting the complex spatial organization of metabolites and offering insights into their biosynthesis.