Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging

[Image: see text] Spatial metabolomics describes the spatially resolved analysis of interconnected pathways, biochemical reactions, and transport processes of small molecules in the spatial context of tissues and cells. However, a broad range of metabolite classes (e.g., steroids) show low intrinsic ionization efficiencies in mass spectrometry imaging (MSI) experiments, thus restricting the spatial characterization of metabolic networks. Additionally, decomposing complex metabolite networks into chemical compound classes and molecular annotations remains a major bottleneck due to the absence of repository-scaled databases. Here, we describe a multimodal mass-spectrometry-based method combining computational metabolome mining tools and high-resolution on-tissue chemical derivatization (OTCD) MSI for the spatially resolved analysis of metabolic networks at the low micrometer scale. Applied to plant toxin sequestration in Danaus plexippus as a model system, we first utilized liquid chromatography (LC)–MS-based molecular networking in combination with artificial intelligence (AI)-driven chemical characterization to facilitate the structural elucidation and molecular identification of 32 different steroidal glycosides for the host-plant Asclepias curassavica. These comprehensive metabolite annotations guided the subsequent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) analysis of cardiac-glycoside sequestration in D. plexippus. We developed a spatial-context-preserving OTCD protocol, which improved cardiac glycoside ion yields by at least 1 order of magnitude compared to results with untreated samples. To illustrate the potential of this method, we visualized previously inaccessible (sub)cellular distributions (2 and 5 μm pixel size) of steroidal glycosides in D. plexippus, thereby providing a novel insight into the sequestration of toxic metabolites and guiding future metabolomics research of other complex sample systems.