Refined on-tissue chemical derivatization expands MALDI-MSI capabilities for spatial metabolomics of mammalian organs using EDC and 4-APEBA

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is widely used in metabolomics because it enables high-resolution spatial characterization of molecular distributions directly from tissue. Nevertheless, comprehensive analysis of small, polar metabolites remains difficult due to limited ionization efficiencies and diverse chemical structures. On-tissue chemical derivatization (OTCD) has emerged as an effective strategy to improve ionization efficiency, sensitivity, and selectivity for these analytes. In this study, we established and systematically optimized an OTCD approach for spatial analysis of carbonyl-and carboxyl-containing metabolites in murine tissues using 4-APEBA in combination with carbodiimide crosslinker EDC. Optimization of reagent concentrations, solvent composition, and spray parameters resulted in robust and reproducible derivatization across multiple tissue types. Application to metastatic liver and lung tissue demonstrated the method’s performance in biologically complex environments. Pronounced metabolic heterogeneity correlated with histopathological features in tumor-rich liver sections, and clear tumor-specific localizations were obtained in lung tissue with only small metastatic foci. Metabolic differences between tumor and adjacent healthy regions were consistent with established cancer metabolism pathways, including glycolytic reprogramming. These findings establish a reliable OTCD workflow for MALDI-MSI-based spatial metabolomics capable of interrogating metabolic heterogeneity in clinically relevant tumor models and demonstrate its value for future investigations of tumor metabolism.