Authors
Cook, A., Deshpande, R., Ellis, A. E., Sheldon, R., Davison, C., Pascoe, J., Bird, S., Beste, D. J., Bailey, M.
Abstract
Single cell metabolomics remains analytically challenging due to the low abundance and chemical diversity of metabolites in individual cells. We have developed complementary microflow HILIC and ion pair LC MS methods to expand metabolite coverage in single macrophages. Ion pair LC MS was applied to single cells for the first time, enabling retention of highly polar and ionic metabolites that elute early under conventional reversed phase conditions. Across Mycobacterium bovis BCG infected, uninfected bystander, and control unexposed THP 1 macrophages, both microflow methods detected significantly more features than a previously reported analytical flow HILIC method. The two microflow methods provided complementary chemical space, together yielding 633 unique named metabolites with MS2 spectra. This depth enabled pathway level interpretation at single cell resolution, revealing infection associated changes in purine-, arginine-, glutathione-, and one carbon folate-associated metabolism. Metabolite-level interrogation indicated shared purine and amino acid changes in both infected and neighbouring macrophages, while revealing a distinct bystander phenotype characterised by elevated glycine and heterogeneous ATP levels. Finally, we demonstrate sequential IP and HILIC analysis of the same single cell, establishing a route toward maximal coverage from individual cells. These results position microflow HILIC and IP LC MS as powerful, orthogonal strategies for advancing single cell metabolomics and unveiling heterogeneity within complex biological microenvironments.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 25 Jun 2026.
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