Comprehensive Metabolite Profiling in Single-Cell Systems via Dual-Modal MALDI-Mass Spectrometry Imaging.

The development of spatial multiomics technologies, particularly matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), has revolutionized our ability to map metabolic processes at single-cell resolution. However, the current techniques face challenges in minimizing matrix interferences and achieving comprehensive metabolite detection across multiple ionization modes. In this study, we present a novel dual-modal MSI workflow that leverages the pairing of 1,5-diaminonaphthalene (DAN) and its hydrochloric salt (DAN-HCl) matrices for sequential detection in positive- and negative-ion modes, respectively. This approach significantly enhanced metabolite coverage, spanning both lipid-based and nonlipid small molecules, while eliminating the need for solvent cleaning steps. Applied to a coculture of cholangiocarcinoma (CCLP1) and hepatic stellate (LX2) cells, the workflow revealed significant metabolic distinctions, including differential accumulation of glycerolipids and energy-related metabolites, highlighting the unique metabolic profiles of each cell type. Additionally, several unidentified metabolites were detected, indicating the potential to discover novel metabolic variations. These findings establish our method as a robust tool for single-cell spatial metabolomics with broad applicability in studying complex cellular interactions and advancing both research and clinical applications.