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

Carbon-13 (13C) NMR provides a powerful approach to non-invasively investigate complex cellular metabolism. However, limitations arise per 13C NMRs reduced sensitivity as compared to proton (1H) NMR, even when using 13C tracer molecules to enrich signal, and particularly when investigating metabolic processes in low volume samples. To overcome these limitations, we developed a 1H NMR method that harnesses 1H NMRs sensitivity with 13C NMRs specificity by capturing 13C NMR information in the 1H spectrum via 13C-1H J-couplings (1H[13C-Jed]). This approach improves 13C signal detection 13.9-fold and incorporates additional 1H NMR information to investigate complex metabolism on a cellular scale. Here, we demonstrate application of 1H[13C- 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 30{micro}L volume, and improved predictions of the pathogens genome scale metabolism. 1H[13C-Jed] NMR provides methodological advancements to investigate dynamic cellular metabolism and its role in complex biologic processes.