Tracking the Metabolic Fate of Exogenous Arachidonic Acid in Ferroptosis Using Dual-Isotope Labeling Lipidomics

Lipid metabolism is implicated in a variety of diseases,
including
cancer, cell death, and inflammation, but lipidomics has proven to
be challenging due to the vast structural diversity over a narrow
range of mass and polarity of lipids. Isotope labeling is often used
in metabolomics studies to follow the metabolism of exogenously added
labeled compounds because they can be differentiated from endogenous
compounds by the mass shift associated with the label. The application
of isotope labeling to lipidomics has also been explored as a method
to track the metabolism of lipids in various disease states. However,
it can be difficult to differentiate a single isotopically labeled
lipid from the rest of the lipidome due to the variety of endogenous
lipids present over the same mass range. Here we report the development
of a dual-isotope deuterium labeling method to track the metabolic
fate of exogenous polyunsaturated fatty acids, e.g., arachidonic acid,
in the context of ferroptosis using hydrophilic interaction–ion
mobility–mass spectrometry (HILIC-IM-MS). Ferroptosis is a
type of cell death that is dependent on lipid peroxidation. The use
of two isotope labels rather than one enables the identification of
labeled species by a signature doublet peak in the resulting mass
spectra. A Python-based software, D-Tracer, was developed to efficiently
extract metabolites with dual-isotope labels. The labeled species
were then identified with LiPydomics based on their retention times,
collision cross section, and m/z values. Changes in exogenous AA incorporation in the absence and
presence of a ferroptosis inducer were elucidated.