Advancing Spatial Lipidomics with MALDI-MS Imaging.

Spatial lipidomics has emerged as a powerful approach for investigating the molecular organization of biological tissues by combining lipid analysis with spatially resolved information. Among the available techniques, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has become a key platform for mapping lipid distributions directly in tissue sections without the need for labeling. This chapter provides a comprehensive overview of recent advances in spatial lipidomics using MALDI-MSI, with particular emphasis on sample preparation strategies, matrix selection and deposition methods, and analytical approaches for lipid identification. Critical steps influencing data quality, including tissue preservation, sectioning, matrix application, and mitigation of ion suppression effects, are discussed in detail. Furthermore, the identification of major lipid classes, such as phospholipids, sphingolipids, neutral lipids, glycolipids, fatty acids, and cholesterol, is addressed through characteristic ionization behaviors and diagnostic fragmentation patterns. Finally, the integration of MALDI-MSI with thin-layer chromatography is presented as a complementary strategy to enhance lipid coverage and reduce ion suppression. Together, these approaches highlight the versatility of MALDI-MSI as a robust tool for advancing spatial lipidomics in biomedical research.