Applications of partial metabolic deuterium oxide labeling to lipidomics

Abstract

In most quantitative mass spectrometry (MS), complete labeling of target analytes with heavy isotopes is designed for simple distinction of isotope-labeled compounds from unlabeled counterparts in a mass spectrum. However, achieving complete metabolic isotope labeling is challenging mainly due to high cost and long time, especially for higher organisms. An alternative method to introduce an isotope to biomolecules is indirect deuterium labeling via deuterium oxide (D2O) administration, which results in strikingly different patterns of mass spectra because of partial isotope enrichment. We have developed novel analytical platforms for turnover rate measurement and relative quantification for lipids on a global scale using metabolic partial D2O labeling. The performance on lipid kinetics measurement of our methods was validated in three different liquid chromatography-MS (LC-MS) setups: MS-only, untargeted MS/MS, and targeted MS/MS. The MS-only scheme consisted of multiple LC-MS runs for quantification of lipid mass isotopomers and an extra LC-MS/MS run for lipid identification. The untargeted MS/MS format utilized multiple data-dependent LC-MS/MS runs for both quantification of lipid mass isotopomers and lipid identification. An in-house software was also developed to streamline the data processing from peak area quantification of mass isotopomers to exponential curve fitting for extracting the turnover rate constant. With HeLa cells cultured in 5% D2O media for 48 hr, we could deduce the species-level turnover rates of 108 and 94 lipids in the MS-only and untargeted MS/MS schemes, respectively, which covers 13 different subclasses and spans 3 orders of magnitude. Furthermore, the targeted MS/MS setup, which performs scheduled LC-MS/MS experiments for some targeted lipids, enabled differential measurement between the turnover rates of the head and tail groups of lipid. The reproducibility of our lipid kinetics measurement was also demonstrated with lipids that commonly detected in both positive and negative ion modes or in two different adduct forms. In order to assess the reproducibility and robustness of our new relative quantification strategy, unlabeled and labeled lipids from HeLa cells were mixed in various mixing ratios. Quantification of equimolar mixtures of HeLa cell lipids revealed high reproducibility and accuracy across three biological and three technical replicates. Two orders of magnitude of dynamic range for relative quantification could also be achieved with HeLa cells variously mixed from 10:1 to 1:10 between unlabeled and labeled lipids. The two critical parameters affecting the accuracy and reliability of the relative quantification were the number of detectable mass isotopomers and the degree of deuterium labeling.