Complementary Single-Cell Microflow HILIC and Ion Pair LC-MS Reveal Bystander Metabolic Effects in a Macrophage Model of Tuberculosis

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. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/733771v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d0d02dorg.highwire.dtl.DTLVardef@11364d5org.highwire.dtl.DTLVardef@40e1a1org.highwire.dtl.DTLVardef@19d24a5_HPS_FORMAT_FIGEXP M_FIG Figure made in BioRender. C_FIG