High-Throughput Fluorescence-Guided Sequential Single-Cell MALDI-ICC Mass Spectrometry.

Lipids are a diverse class of biomolecules essential for brain function, yet their cell-type-specific distributions remain underexplored, presenting significant knowledge gaps in the era of single-cell biology. Traditional bulk measurements provide valuable insights into lipid composition across brain regions but lack the resolution to distinguish lipid profiles at the single-cell level. To address this, we introduce fluorescence-guided sequential single-cell mass spectrometry (SSMS), an automated workflow combining untargeted lipid profiling with antibody-targeted protein detection via photocleavable mass tags, enabling neurolipidomic classification of cell types and cell states. We applied this approach to rodent hippocampal cells, analyzing over a thousand single cells and annotating more than a hundred lipid species with complementary liquid chromatography-mass spectrometry (LC-MS/MS) measurements. Our findings show that phosphatidylcholine (PC) species are predominantly enriched in oligodendrocytes and neurons compared to astrocytes, while hexosylceramide (HexCer) species are differentially expressed across these cell types. Furthermore, neuronal state analysis revealed an enrichment of phosphatidylethanolamines (PEs) in presynaptic neurons, while nonpresynaptic neurons exhibited a more diverse lipid composition, including HexCer, PC, sphingomyelin, triacylglycerol, and PE. Our findings provide new insights into brain lipid heterogeneity with cell-type and cell-state specificity and extend-capabilities of next-generation single-cell mass spectrometry to map brain biochemistry.