Metabolic Heavy Water Labeling for 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 heavy water (2H2O) administration, which results in strikingly different patterns of mass spectra because of partial isotope enrichment. We have developed novel analytical platforms for relative quantification for lipids on a global scale using metabolic partial 2H2O labeling. 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. Finally, an in-house software written in C++ and python was developed to streamline the data processing from calculating the peak area of extracted ion chromatogram of each mass isotopomer of lipid ions to the relative quantification of equimolr mixtures of unlabeled and labeled lipids.