Mass Spectrometry Imaging (MSI) enables spatial mapping of metabolites but often lacks in-situ structural confirmation. To address this, we validated a workflow combining histological staining, Desorption Electrospray Ionization (DESI)-MSI for spatial metabolic mapping and Liquid Extraction Surface Analysis (LESA)-MS2 for structural identification. The integrated approach allows comprehensive in situ detection and structural confirmation of metabolites using MS2 spectra, eliminating ambiguity and allowing confident molecular identification. We applied this workflow to full body sections of adult zebrafish (Danio rerio) fed a customized high-fat, high-cholesterol diet (HFD). Our multimodal imaging approach showed high analytical reproducibility during validation across tissues and highlighted the tissue specific lipidome signature. Unsupervised clustering of DESI-MSI data accurately identified adipose depots based solely on their lipid signature which were then confirmed by histology. Receiver operating characteristic (ROC) analysis and subsequent LESA-MS2 molecular confirmation led to identification of 52 lipids in adipose tissue, discriminating from non-adipose regions and included Di- and Triglycerides (DAGs and TAGs), free fatty acids (FFAs) and oxidized FFAs. This study establishes an optimized spatial lipidomics workflow and provides the first spatially resolved lipidomic profile of zebrafish adipose tissue. The integrated approach is broadly applicable to scenarios where sample material is limited, such as clinical biopsies or organoid models.
Isfort et al. (Thu,) studied this question.