HT SpaceM: A High-Throughput and Reproducible Method for Small-Molecule Single-Cell Metabolomics

Delafiori, J.; Shahraz, M.; Eisenbarth, A.; Hilsenstein, V.; Drotleff, B.; Bailoni, A.; Wadie, B.; Ekelöf, M.; Mattausch, A.; Alexandrov, T.

doi:10.1101/2024.10.24.620114

bioRxiv · 10.1101/2024.10.24.620114

HT SpaceM: A High-Throughput and Reproducible Method for Small-Molecule Single-Cell Metabolomics

Delafiori, J.·Shahraz, M.·Eisenbarth, A.·Hilsenstein, V.·Drotleff, B.·Bailoni, A.·Wadie, B.·Ekelöf, M.·Mattausch, A.·Alexandrov, T.

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Abstract

Single-cell metabolomics promises to resolve metabolic cellular heterogeneity, yet current methods struggle with detecting small molecules, throughput, and reproducibility. Addressing these gaps, we developed HT SpaceM, a high-throughput single-cell metabolomics method with novel cell preparation, custom glass slides, small-molecule MALDI imaging mass spectrometry protocol, and batch processing. We propose a unified framework covering essential data analysis steps including quality control, characterization, differential analysis, structural validation and functional analysis. Interrogating human HeLa and mouse NIH3T3 cells, we detected 73 diverse small-molecule metabolites validated by bulk LC-MS/MS, achieving high reproducibility across wells and slides. Interrogating nine NCI-60 cancer cells and HeLa, we identified cell-type markers in small subpopulations. Functional analysis revealed overrepresented metabolic pathways, co-abundant metabolites, and metabolic hubs. We demonstrate the ability of SCM to analyze over 120,000 cells from over 112 samples, and provide guidance to interpret single-cell metabolic heterogeneity, revealing metabolic insights beyond population averages.

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Delafiori, J., Shahraz, M., Eisenbarth, A., Hilsenstein, V., Drotleff, B., Bailoni, A., Wadie, B., Ekelöf, M., Mattausch, A., Alexandrov, T.. 2024-10-25. HT SpaceM: A High-Throughput and Reproducible Method for Small-Molecule Single-Cell Metabolomics. https://doi.org/10.1101/2024.10.24.620114

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