A Robust IsotopeRatio LC–MS/MS Workflow forHigh-Throughput Metabolic Profiling of Bacteria

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 13 C-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 13 C-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. Abstract Figure