¹⁵N‑NMR-Based Approach for Amino Acids-Based ¹³C‑Metabolic Flux Analysis of Metabolism

NMR analysis of the isotope incorporation in amino acids can be used to derive information about the topology and operation of cellular metabolism. Although traditionally performed by ¹H and/or ¹³C NMR, we present here novel experiments that exploit the ¹⁵N nucleus to derive the same information with increased efficiency. Combined with a novel Hα-¹³CO experiment, we increase the coverage of the isotopic space that can be probed by obtaining the complete distribution of isotopic species for the first two carbons of amino acids in cellular biomass hydrolysates. Our approach was evaluated using as reference material a biologically produced sample containing ¹⁵N-labeled metabolites with fully predictable ¹³C-labeling patterns. Results show excellent agreement between measured and expected isotopomer abundances for the different NMR experiments, with an accuracy and precision within 1%. We also demonstrate how these experiments can give detailed information about metabolic fluxes depending on the expression level of a critical enzyme. Hence, exploiting the ¹⁵N labeling of a cellular sample accelerates subsequent analysis of the hydrolyzed biomass and increases the coverage of isotopomers that can be quantified, making it a promising tool to increase the throughput and the resolution of ¹³C-fluxomics studies

Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments

Stable-isotope labeling experiments (ILEs) are widely used to investigate the topology and operation of metabolic networks. The quality of isotopic data collected in ILEs is of utmost importance to ensure reliable biological interpretations, but current evaluation approaches are limited due to a lack of suitable reference material and relevant evaluation criteria. In this work, we present a complete methodology to evaluate mass spectrometry (MS) methods used for quantitative isotopic studies of metabolic systems. This methodology, based on a biological sample containing metabolites with controlled labeling patterns, exploits different quality metrics specific to isotopic analyses (accuracy and precision of isotopologue masses, abundances, and mass shifts and isotopic working range). We applied this methodology to evaluate a novel LC-MS method for the analysis of amino acids, which was tested on high resolution (Orbitrap operating in full scan mode) and low resolution (triple quadrupole operating in multiple reaction monitoring mode) mass spectrometers. Results show excellent accuracy and precision over a large working range and revealed matrix-specific as well as mode-specific characteristics. The proposed methodology can identify reliable (and unreliable) isotopic data in an easy and straightforward way and efficiently supports the identification of sources of systematic biases as well as of the main factors that influence the overall accuracy and precision of measurements. This approach is generic and can be used to validate isotopic analyses on different matrices, analytical platforms, labeled elements, or classes of metabolites. It is expected to strengthen the reliability of isotopic measurements and thereby the biological value of ILEs

Improved Isotopic Profiling by Pure Shift Heteronuclear 2D <i>J</i>‑Resolved NMR Spectroscopy

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 <i>J</i>-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