OzFAD: Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome

Abstract

Fatty acid isomers are responsible for an under-reported lipidome diversity across all kingdoms of life. Isomers of unsaturated fatty acid are often masked in contemporary analysis by incomplete separation and the absence of sufficiently diagnostic methods for structure elucidation. Here, we introduce a comprehensive workflow to discover new unsaturated fatty acids through coupling liquid chromatography and mass spectrometry with gas-phase ozonolysis of double bonds. The workflow encompasses semi-automated data analysis and enables de novo identification in complex media including human plasma, cancer cell lines and human sebaceous wax (i.e., vernix caseosa). The targeted analysis including ozonolysis enables structural assignment over a dynamic range of five orders of magnitude, even in instances of incomplete chromatographic separation. Thereby we expand the number of identified plasma fatty acids two-fold, including non-methylene interrupted fatty acids. Detection, without prior knowledge, allows discovery of non-canonical double bond positions. Changes in relative isomer abundances reflect underlying perturbations in lipid metabolism.

Establishing carbon–carbon double bond position and configuration in unsaturated fatty acids by gas-phase infrared spectroscopy

“…The composite spectrum regression yielded a ratio of approximately 3 : 1 9 Z : 9 E . As a control experiment, liquid chromatography-ozone-induced dissociation (LC-OzID-MS) 39 tandem mass spectrometry was used to confirm isomer assignment and relative abundance by integration of the chromatogram (Fig. 3b).…”

“…The estimated relative error of relative quantities of octadecenoic acids determined by LC-OzID-MS is ±20% (based on the triplicate measurement of a pooled human plasma – NIST 1950 – standard reference material, total hydrolysed fatty acid content, average coefficient of variation of relative quantities of all detected octadecenoic acids). 39 A determined 10% relative quantity, for example, should thus be read as 8–12%. Overall, the analysis of two hydrolysed lipid extracts of biological origin showcases the power of gas-phase IR spectroscopy for the analysis of isomeric mixtures, even in complex matrices.…”

MS-DIAL 5 multimodal mass spectrometry data mining unveils lipidome complexities

“…This allows the characterization of lipid structures at the molecular species level, characterizing lipid subclasses in addition to carbon and double bond numbers in the individual acyl chains 2 . In addition, advanced techniques such as electron-based method 3 , Paterno-Buchi reaction 4 , ultraviolet photodissociation 5 , and ozone- or hydroxyl-radical reactions 6,7 offer deeper insights into lipid structures by annotating the sn -position and double bond (C=C) locations. Moreover, spatial lipidomics, such as matrix-assisted laser desorption ionization (MALDI) coupled with MS, facilitate the determination of lipid molecule localizations 8 .…”

In-Depth Characterization of Sphingoid Bases via Radical-Directed Dissociation Tandem Mass Spectrometry

“…This aspect also makes RDD less sensitive in pinpointing C�C location as compared to those C�C specific fragmentation methods, including PB-MS/MS, OzID, etc. As lipids containing branched and unsaturated chains are not uncommon, 44 strategies that can harness the analytical advantages of both RDD and C�C specific fragmentation methods are highly desirable for future development.…”