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

• Published in: Rapid communications in mass spectrometry 2014-02, Vol.28 (3), p.239-244
• Main Authors: Herath, Kithsiri B., Zhong, Wendy, Yang, Jiong, Mahsut, Ablatt, Rohm, Rory J., Shah, Vinit, Castro-Perez, Jose, Zhou, Haihong, Attygalle, Athula B., Kang, Ling, Singh, Sheo, Johns, Douglas G., Cleary, Michele A., Hubbard, Brian K., Previs, Stephen F., Roddy, Thomas P.
• Format: Article
• Language: English
• Subjects: Animals; Cercopithecus aethiops; Deuterium - analysis; Deuterium - chemistry; Deuterium - metabolism; Deuterium Oxide - administration & dosage; Fatty Acids - chemistry; Fatty Acids - metabolism; Female; Isotope Labeling - methods; Linear Models; Macaca mulatta; Male; Mass Spectrometry - methods; Peptides - chemistry; Peptides - metabolism
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.6776

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.