Improved Isotopic Profiling by Pure Shift Heteronuclear 2D J-Resolved NMR Spectroscopy

Abstract

Quantitative information on the carbon isotope content of metabolites is essential for flux analysis. Whereas this information is in principle present in proton NMR spectra through both direct and long-range heteronuclear coupling constants, spectral overlap and homonuclear coupling constants both hinder its extraction. We demonstrate here how pure shift 2D J-resolved NMR spectroscopy can simultaneously remove the homonuclear couplings and separate the chemical shift information from the heteronuclear coupling patterns. We demonstrate the power of this method on cell lysates from different bacterial cultures and investigate in detail the branched chain amino acid biosynthesis.

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

“…As a result, even though the sample is a bacterial lysate containing only isolated metabolites (including amino acids) with sharp signals for every isotopomer if one can remove the homonuclear couplings, the composite peaks appear as broad humps. This is confirmed when we extract from the heteronuclear pure shift J‐resolved spectrum the pure chemical shift of the 12 C‐linked methyl protons in the absence of any heteronuclear or homonuclear couplings, where indeed we do observe the methyls as sharp peaks. We can thereby observe at 800 MHz a 9‐Hz difference between the Ile Hδ methyl signal and the closest Leu Hδ neighbor.…”

“…The spectrum at 1.1 GHz was acquired on the instrument at Bruker (Fällanden, Switzerland) with a 3‐mm TCI probe, on the same PT sample as was used for the spectral recording at 500 and 800 MHz. The pure shift heteronuclear J‐resolved spectra were acquired at 500 MHz and 800 MHz as described . The SAPPHIRE spectrum was acquired with 2kx8x64 points and was processed as proposed by the authors .…”

“…We recently described a method combining heteronuclear J-resolved NMR spectroscopy with pure-shift methodology to detect the level of enrichment for each site in the BCAAs in a single experiment. [7] Using 13 Clabeled glucose and unlabeled Thr as sole carbon sources for the cell growth, we could show in a single experiment that the C β -C γ1 block of isoleucine indeed comes from the 13 C-labeled glucose, whereas all other carbons find their origin in the unlabeled Thr. However, the pure-shift character of this experiment leads to a decreased sensitivity [8][9][10] and further increases the dimensionality of the experiment from a 2D experiment for the heteronuclear J-resolved pulse sequence to a 3D experiment.…”

“…We recently described a method combining heteronuclear J‐resolved NMR spectroscopy with pure‐shift methodology to detect the level of enrichment for each site in the BCAAs in a single experiment . Using 13 C‐labeled glucose and unlabeled Thr as sole carbon sources for the cell growth, we could show in a single experiment that the C β –C γ1 block of isoleucine indeed comes from the 13 C‐labeled glucose, whereas all other carbons find their origin in the unlabeled Thr.…”

“…However, the pure-shift character of this experiment leads to a decreased sensitivity [8][9][10] and further increases the dimensionality of the experiment from a 2D experiment for the heteronuclear J-resolved pulse sequence to a 3D experiment. [7] As a result, the experiment requires at least a night. This requirement becomes an important issue when multiple samples have to be analyzed, even though the amount of material or the concentration of the BCAAs in the bacterial hydrolysate is not directly the problem.…”