Determination of low levels of 2H-labeling using high-resolution mass spectrometry: Application in studies of lipid flux and beyond.

RATIONALE The ability to measure low levels of ²H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering ²H₂O and then measuring the incorporation of ²H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. ¹³C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from ¹³C) whereas the latter is not suitable for routine high-throughput analyses. METHODS We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the ²H-enrichment of fatty acids and peptides. RESULTS In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the ²H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the ²H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS High-resolution mass spectrometry can be used to measure low levels of ²H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on ²H-labeled tracers, e.g. ²H₂O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]