Spatial metabolomic profiling of ripening pepper fruit (Capsicum annuum L.) by MALDI-MSI: Decoding metabolic heterogeneity and its role in quality attribute regulation.

To decipher the metabolic regulation of pepper fruit (Capsicum annuum L.) quality, tissue-scale matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was employed to systematically map 19 key metabolites across four developmental stages at a spatial resolution of 50 μm. Imaging revealed distinct spatiotemporal dynamics: capsaicinoids exhibited a localized "bimodal accumulation" in the placenta, while capsanthin precursors shifted from a uniform distribution to specific enrichment in the pericarp. Notably, a synthesis-distribution decoupling was observed in ascorbic acid metabolism, suggesting potential inter-tissue redistribution. Integration with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the absolute quantification of four core metabolites (n = 3) provided complementary insights, distinguishing continuous bulk accumulation from dynamic, non-linear local metabolic activity. This study constructs a tissue-scale spatial metabolomic atlas, highlighting the synergistic value of combining spatial imaging with bulk quantification to unravel metabolic heterogeneity in food matrices.