Mass‐Guided Single‐Cell MALDI Imaging of Low‐Mass Metabolites Reveals Cellular Activation Markers

Abstract 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. However, cell‐targeting and metabolome‐preserving methods for analysis of low mass, hydrophilic metabolites (<200 Da) in large cell populations are lacking. Here, the PRISM‐MS ( PR escan I maging for S mall M olecule – M ass S pectrometry) mass‐guided MSI workflow is presented, which enables space‐efficient single cell lipid and metabolite analysis. In conjunction with 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, 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 can serve as a standard method in single cell metabolomics, given its capability to characterize larger cell populations and low‐mass metabolites.