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bioRxiv · 10.1101/2025.08.08.669109

A Robust Isotope Ratio LC-MS/MS Workflow for High-Throughput Metabolic Profiling of Bacteria

Rocha, C.·Pinto, S. P.·Jensen, S. I.·Nielsen, L. K.·Donati, S.
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Abstract

Stable isotope dilution mass spectrometry (IDMS) has become a cornerstone of quantitative metabolomics, enabling accurate intracellular metabolite quantification across a range of biological systems. However, the broader adoption of IDMS in high-throughput studies remains limited by the high costs of commercially available 13C-labeled internal standards (IS), labor-intensive in-house IS production, and the narrow applicability of existing methods to different organisms. Here, we present a robust and scalable IDMS-based LC-MS/MS workflow for high-throughput metabolic profiling of diverse bacteria. The analytical method couples ion-pairing liquid chromatography with multiple reaction monitoring (MRM) to quantify 96 intracellular metabolites in under 16 minutes, with an average RSD of 21%. We developed a protocol for large-scale production of high-quality 13C-labeled IS, considerably lowering the cost for high-throughput IDMS studies. We then applied the workflow to 5 bacterial species in different cultivation conditions. This work provides a versatile platform for microbial metabolomics, supporting systems biology and data-driven metabolic engineering at scale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/669109v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f3e9e9org.highwire.dtl.DTLVardef@81eb0eorg.highwire.dtl.DTLVardef@16efe09org.highwire.dtl.DTLVardef@1e83a06_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Rocha, C., Pinto, S. P., Jensen, S. I., Nielsen, L. K., Donati, S.. 2025-08-11. A Robust Isotope Ratio LC-MS/MS Workflow for High-Throughput Metabolic Profiling of Bacteria. https://doi.org/10.1101/2025.08.08.669109

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