Fatty acid isomers are responsible for an under-reported lipidome diversity across all kingdoms of life. Isomers of unsaturated fatty acids 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 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 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.

• MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• lipidomika MeSH
• mastné kyseliny * chemie   MeSH
• nenasycené mastné kyseliny chemie   MeSH
• ozon * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mastné kyseliny * MeSH
• nenasycené mastné kyseliny MeSH
• ozon * MeSH

The specific positions of carbon-carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography-mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N-(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon-carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa. This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways.

• Klíčová slova
• Fatty Acids, Lipids, ozone-induced dissociation, vernix caseosa,
• MeSH
• lidé MeSH
• mastné kyseliny analýza   chemie   MeSH
• ozon chemie   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• triglyceridy analýza   chemie   MeSH
• vernix caseosa chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mastné kyseliny MeSH
• ozon MeSH
• triglyceridy MeSH