Deconvoluting Metabolomic Flux in Living Cells with Real-time ¹³ C J-coupling-edited Proton High-Resolution Magic Angle Spinning (HRMAS) NMR

Carbon-13 ( ¹³ C) NMR provides a powerful approach to non-invasively investigate complex cellular metabolism. However, limitations arise per ¹³ C NMR’s reduced sensitivity as compared to proton ( ¹ H) NMR, even when using ¹³ C tracer molecules to enrich signal, and particularly when investigating metabolic processes in low volume samples. To overcome these limitations, we developed a ¹ H NMR method that harnesses ¹ H NMR’s sensitivity with ¹³ C NMR’s specificity by capturing ¹³ C NMR information in the ¹ H spectrum via ¹³ C- ¹ H J-couplings ( ¹ H[ ¹³ C-Jed]). This approach improves ¹³ C signal detection 13.9-fold and incorporates additional ¹ H NMR information to investigate complex metabolism on a cellular scale. Here, we demonstrate application of ¹ H[ ¹³ C- Jed] NMR to deconvolute complex cellular metabolism in the anaerobic pathogen Clostridioides difficile . Analyses enabled precise tracking of branching metabolic reactions under anoxic and reducing conditions over time in a 30µL volume, and improved predictions of the pathogen’s genome scale metabolism. ¹ H[ ¹³ C-Jed] NMR provides methodological advancements to investigate dynamic cellular metabolism and its role in complex biologic processes.