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

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 (ISs), 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 the high-throughput profiling of primary metabolism in diverse bacteria. The analytical method couples ion-pairing liquid chromatography with multiple reaction monitoring (MRM) to quantify 96 intracellular metabolites in under 16 min, with an average RSD of 21%. We developed a protocol for large-scale production of high-quality 13C-labeled IS, considerably lowering the costs and labor necessary to perform high-throughput IDMS studies. We then demonstrated the applicability of the same workflow by performing relative quantification of 5 diverse 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.