biostudies-otherUnknownJeany DelafioriEuropean Research Council (Consolidator grant)German Research FoundationEuropean Research Council (Proof-of-Concept grant)Swiss National Science FoundationMichael J. Fox FoundationHomo sapiens (human)Mus musculus (mouse)https://www.ebi.ac.uk/biostudies/studies/S-BSST2127biostudies-otherAI 186/1-1Sinergia grant PROMETEX773089101101077Bishoy WadieAndreas EisenbarthAlexander MattauschMåns EkelöfAlberto BailoniJeany DelafioriTheodore AlexandrovMohammed ShahrazBernhard DrotleffVolker HilsensteinfalseHT SpaceM: A High-Throughput and Reproducible Method for Small-Molecule Single-Cell MetabolomicsSingle-cell metabolomics (SCM) promises to reveal metabolism in its complexity and heterogeneity, yet current methods struggle with detecting small-molecule metabolites, throughput, and reproducibility. Addressing these gaps, we developed HT SpaceM, a high-throughput SCM method combining cell preparation in custom glass slides, small-molecule MALDI imaging mass spectrometry, and batch processing. We propose a unified framework covering quality control, characterization, structural validation, differential and functional analysis. Profiling HeLa and NIH3T3 cells, we detected 73 small-molecule metabolites validated by bulk LC-MS/MS, achieving high reproducibility and single-cell resolution. Interrogating nine NCI-60 cancer cell lines and HeLa, we identified cell-type markers in subpopulation2025-08-28T00:00:00Z2026-04-13T15:03:35.215Z2025-07-26T19:04:57.061ZS-BSST2127