Real-time 13C J-coupling-edited Proton High-Resolution Magic Angle Spinning (HRMAS) MRS of Live Cells

Motivation: Real-time biochemical changes at the cellular-level can be monitored using 13C-labelled substrates via 13C-MRS. However, a small sample volume, spectral overlap and low sensitivity of MRS make long-term monitoring of targeted metabolic pathways challenging. Goal(s): Use 1H-13C-J-coupling (1H[13C-Jed]) to observe protons attached to 13C-labelled substrates, enabling targeted metabolic process monitoring, in vivo, with increased sensitivity. Approach: Pathogenic Clostridioides difficile was grown in 13C-media (13C-glucose and 13C-threonine). 13C-HRMAS MRS of 100, 000 cells (20 μL), was used to monitor cellular metabolism for >48 hours. Results: Downstream metabolic breakdown products and a 13.9X improvement in S/N by 1H[13C-Jed], compared to standard 13C-MRS, was observed. Impact: 1H-13C-J-coupling (1H[13C-Jed]) HRMAS was used to follow real-time targeted metabolism of 13C-glucose and 13C-threonine in 100,000 C. diff. cells (20 μL) using 13C-labelled substrates, for >48 hours. A 13.9x improvement in S/N in 1H[13C-Jed relative to 13C-MRS was observed.