Stable isotope labeling in metabolomic studies

Metabolomic pathways in biological systems can be explored using stable isotope labeling (the most common isotopes used for labeling are 2H, 13C, and 15N).  In these experiments cells, plants, or living organisms are fed with an isotopically labeled precursor following the determination of the ratios between natural and enriched isotopes in various metabolites.

The high resolving power of a mass spectrometer is essential for the accurate determination of isotope markers incorporated into the moiety of an organic molecule, particularly at lower levels of enrichment. That is because other natural stable but minor isotopes may interfere with the isotope used for labeling study. The results of the analyses demonstrated herein could be achieved only using the resolving power of the mass spectrometer at ~150,000-250,000.

Abundances of some minor isotopes in nature (relative to the majors) are:  2H - 0.012%, 13C - 1.082%, 15N - 0.369%, 18O - 0.205%, 

Instrument: Isotope composition studies were carried out using the Q-Exactive Plus hybrid FT mass spectrometer coupled with either Dionex Ultimate 3000 RS UPLC or Integrion ion-chromatograph (Thermo Scientific). The mass spectrometer was operated at the maximal available resolving power.

Study 1. There was investigated the consumption of glucose (energy) in mice in this study. For that purpose, a mouse was injected with a certain amount of glucose-D2, following the determination of the ratio between the natural and labeled glucose at defined periods of time. 

The mass spectra of the deprotonated glucose were simulated at different resolving powers for demonstration purposes. From the examples of the three mass spectra below, it can be learned that the correct isotope analysis of the M+2 mass can be correctly measured only at the resolving power of 150,000 or above.

The ion M is composed solely of the major isotopes i.e.,  1H, 12C, and 16O.

The ion M+1 is due to the 13C isotope, - (1.082 × 6 (number of carbons in glucose) = 6.5%.

The ion M+2 is composed of 18O (0.205× 6 = 1.23%) and a small amount of 13C2. Remember, we are going to measure the glucose-D2 i.e., M+2.

Study 3. Investigation of nitrogen assimilation metabolic pathways in plants under high light conditions. The Arabidopsis plants were nourished with K15NO3 under different light conditions following the LC-HRMS analysis of free amino acids. The compounds were separated on HILIC-Z (Agilent) column.