High-Throughput Measurement of Lipid Turnover Rates Using Partial Metabolic Heavy Water Labeling

Abstract

Novel analytical platforms for high-throughput determination of lipid turnover in vivo have been developed based on partial metabolic HO labeling. The performance on lipid kinetics measurement of our methods was validated in three different liquid chromatography-mass spectrometry (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% HO media for 48 h, 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.

Metabolically-Incorporated Deuterium in Myelin Localized by Neutron Diffraction and Identified by Mass Spectrometry

“…Thus, the convergence of lipidomics and metabolomics can be anticipated to provide extraordinary insight into myelinomics. An indication of the analytical and quantitative power of this approach has been demonstrated using HeLa cells [64,65]. In short, we anticipate that our correlated structural and biochemical study will promote a broad, comprehensive approach that is informative about structure, function, and metabolism in both health and disease.…”

“…Thus, the convergence of lipidomics and metabolomics can be anticipated to provide extraordinary insight into myelinomics. The analytic and quantitative power of this approach has been demonstrated using HeLa cells (Goh et al, 2018; Kim et al, 2019). Further, in the experimental paradigm we used here for stable isotope incorporation via D 2 O-spiked drinking water, not only was myelin labeled, but one can surmise a priori that all tissues in the mice were deuterated, and differentially for the pups vs. the dam.…”

Partial Isotope Profiles Are Sufficient for Protein Turnover Analysis Using Closed-Form Equations of Mass Isotopomer Dynamics

“…However, the equations are general. They can be used for other atom-based labeling agents, ,, such as 15 N and 12 C, or for other biomolecules such as lipids …”

“…They can be used for other atom-based labeling agents, 4,10,11 such as 15 N and 12 C, or for other biomolecules such as lipids. 12 1.1. Data Description.…”

Timepoint Selection Strategy for In Vivo Proteome Dynamics from Heavy Water Metabolic Labeling and LC–MS

“…The time course of the label incorporation is modeled to extract the degradation rate constants of proteins. − Bioinformatics tools ,− have been developed for processing high-throughput datasets. A similar approach has been used to analyze lipidome dynamics as well . In the time-course models, dense sampling provides accurate rate constant estimations.…”