Increasing field strength versus advanced isotope labeling for NMR‐based fluxomics

Abstract

Nuclear magnetic resonance (NMR)‐based fluxomics seeks to measure the incorporation of isotope labels in selected metabolites to follow kinetically the synthesis of the latter. It can however equally be used to understand the biosynthetic origin of the same metabolites. We investigate here different NMR approaches to optimize such experiments in terms of resolution and time requirement. Using the isoleucine biosynthesis as an example, we explore the use of different field strengths ranging from 500 MHz to 1.1 GHz. Because of the different field dependence of chemical shift and heteronuclear J couplings, the spectra change at different field strengths. We equally explore the approach to silence the leucine/valine methyl signals through the use of a suitable deuterated precursor, thereby allowing selective observation of the Ile 13C labeling pattern. Combining both approaches, we arrive at an efficient procedure for the NMR‐based exploration of Ile biosynthesis.

Improved NMR Detection of Phospho-Metabolites in a Complex Mixture

“…However, the experimental conditions varying slightly between our extract and the standard conditions used to record the database 3PG entries, chemical shift values are close but not identical (Figure S7), and this will be a recurrent issue for database mining with spectra of phospo-metabolites. Another potentially confounding factor is that the J-coupling values when expressed in ppm become field-dependent, 58 and can thereby distort the apparent pattern. The Birmingham Metabolite library 36 or SpinCouple 25 avoids this issue by using tilted spectra and assign thereby all lines of a multiplet to a single chemical shift.…”