Fast Single-Cell MALDI Imaging of Low-Mass Metabolites Reveals Cellular Activation Markers

Single-cell MALDI mass spectrometry imaging (MSI) of lipids and metabolites >200 Da has recently come to the forefront of biomedical research and chemical biology, but fast metabolome-preserving methods without paraformaldehyde fixation for analysis of low mass, hydrophilic metabolites (<200 Da) in large cell populations are lacking. Introducing giant unilamellar vesicles (GUVs) as MSI ground truth for cell-sized objects and Monte Carlo reference-based consensus clustering for data-dependent identification of cell subpopulations. The PRISM-MS ( PR escan I maging for S mall M olecule - M ass S pectrometry) dual-scan MSI workflow is presented, enabling space-efficient and therefore faster lipid analysis in single GUVs and cells. Beyond lipids, PRISM-MS enables MSI and on-cell MS2-based identification of low-mass metabolites like amino acids or Krebs cycle intermediates involved in stimulus-dependent cell activation. The utility of PRISM-MS is demonstrated through the characterization of complex metabolome changes in lipopolysaccharide (LPS)-stimulated microglial cells and human-induced pluripotent stem cell-derived microglia. Translation of single cell results to endogenous microglia in organotypic hippocampal slice cultures indicates that LPS-activation involves changes of the itaconate-to-taurine ratio and alterations in neuron-to-glia glutamine-glutamate shuttling. The data suggests that PRISM-MS could serve as a standard method in single cell metabolomics, given its capability to characterize larger cell populations and low-mass metabolites.