Mapping CC Positions in Branched Fatty Acid Esters of Hydroxy Fatty Acids by Oxygen Attachment Dissociation Mass Spectrometry Coupled with Chemical Labeling

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) represent an important class of endogenous lipids with demonstrated anti-inflammatory and antidiabetic functions. However, comprehensive structural characterization, particularly the unambiguous localization of carbon–carbon double (CC) bonds in unsaturated FAHFAs, remains analytically challenging due to structural diversity, low abundance, and the lack of reference standards. Herein, we introduce a novel mass-spectrometry-based strategy termed DISCO-MS (derivatization-initiated source and collision OAD mass spectrometry) for high-confidence FAHFA annotation. The method combines oxygen attachment dissociation (OAD) with in-source fragmentation (ISF) in a hyphenated workflow, enabling the simultaneous determination of CC bond positions, fatty acyl chain lengths, and ester bond information in a single run. A Python-based multimass spectral generator was developed to predict and curate a comprehensive database containing 58,506 MS/MS spectra covering 19,502 putative FAHFA species. The utility of DISCO-MS was demonstrated through the analysis of six custom-synthesized unsaturated FAHFA standards and complex biological extracts, revealing a previously unresolved structural diversity. This integrated approach provides a promising platform for accelerating the discovery and functional study of FAHFAs in metabolic and nutritional research.