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

RATIONALEThe ability to measure low levels of H-2-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (H2O)-H-2 and then measuring the incorporation of H-2 into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. C-13. 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 C-13) whereas the latter is not suitable for routine high-throughput analyses. METHODSWe have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the H-2-enrichment of fatty acids and peptides. RESULTSIn contrast to IRMS, which requires 30min per analysis, it is possible to measure the H-2-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in 3min per sample. In addition, Qq-FT-ICR MS enabled measurements of the H-2-enrichment of peptides (which is not possible using IRMS). CONCLUSIONSHigh-resolution mass spectrometry can be used to measure low levels of H-2-labeling so we expect that this approach will enhance studies of metabolic flux that rely on H-2-labeled tracers, e.g. (H2O)-H-2. 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 (c) 2013 John Wiley & Sons, Ltd.