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.

Centre for Data Science Queensland University of Technology Brisbane QLD 4000 Australia

Centre for Materials Science Queensland University of Technology Brisbane QLD 4000 Australia

Department of Analytical Chemistry Faculty of Science Charles University Prague 2 Czech Republic

Faculty of Science Medicine and Health School of Chemistry and Molecular Bioscience Wollongong NSW Australia

Institute of Clinical Chemistry Inselspital Bern University Hospital 3010 Bern Switzerland

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo náměstí 542 2 16600 Prague Czech Republic

School of Biomedical Sciences Faculty of Health Queensland University of Technology Translational Research Institute Brisbane QLD 4102 Australia

School of Chemistry and Physics Queensland University of Technology Brisbane QLD 4000 Australia

South Australian Health and Medical Research Institute Adelaide SA Australia

South Australian Immunogenomics Cancer Institute and Freemasons Centre for Male Health and Wellbeing University of Adelaide Adelaide SA Australia

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Perez de Souza L, Alseekh S, Scossa F, Fernie AR. Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research. Nat. Methods. 2021;18:733–746. doi: 10.1038/s41592-021-01116-4. PubMed DOI

Gault J, et al. Combining native and ‘omics’ mass spectrometry to identify endogenous ligands bound to membrane proteins. Nat. Methods. 2020;17:505–508. doi: 10.1038/s41592-020-0821-0. PubMed DOI PMC

Rustam YH, Reid GE. Analytical challenges and recent advances in mass spectrometry based lipidomics. Anal. Chem. 2018;90:374–397. doi: 10.1021/acs.analchem.7b04836. PubMed DOI

Slatter DA, et al. Mapping the human platelet lipidome reveals cytosolic phospholipase A2 as a regulator of mitochondrial bioenergetics during activation. Cell Metab. 2016;23:930–944. doi: 10.1016/j.cmet.2016.04.001. PubMed DOI PMC

Harayama T, Riezman H. Understanding the diversity of membrane lipid composition. Nat. Rev. Mol. Cell Biol. 2018;19:281–296. doi: 10.1038/nrm.2017.138. PubMed DOI

Feng G, et al. Dual-resolving of positional and geometric isomers of C=C bonds via bifunctional photocycloaddition-photoisomerization reaction system. Nat. Commun. 2022;13:2652. doi: 10.1038/s41467-022-30249-z. PubMed DOI PMC

Quehenberger O, Armando AM, Dennis EA. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography–mass spectrometry. Biochim. Biophys. Acta. 2011;1811:648–656. doi: 10.1016/j.bbalip.2011.07.006. PubMed DOI PMC

Mitchell TW, Pham H, Thomas MC, Blanksby SJ. Identification of double bond position in lipids: from GC to OzID. J. Chromatogr. B. 2009;877:2722–2735. doi: 10.1016/j.jchromb.2009.01.017. PubMed DOI

Zehethofer N, Pinto DM. Recent developments in tandem mass spectrometry for lipidomic analysis. Anal. Chim. Acta. 2008;627:62–70. doi: 10.1016/j.aca.2008.06.045. PubMed DOI

Brenna JT. Fatty acid analysis by high resolution gas chromatography and mass spectrometry for clinical and experimental applications. Curr. Opin. Clin. Nutr. Metab. Care. 2013;16:548–554. doi: 10.1097/MCO.0b013e328363bc0a. PubMed DOI

Christie WW. Gas chromatography-mass spectrometry methods for structural analysis of fatty acids. Lipids. 1998;33:343–353. doi: 10.1007/s11745-998-0214-x. PubMed DOI

Campbell JL, Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics. Anal. Chem. 2015;87:5837–5845. doi: 10.1021/acs.analchem.5b01460. PubMed DOI

Baba T, et al. Dissociation of biomolecules by an intense low-energy electron beam in a high sensitivity time-of-flight mass spectrometer. J. Am. Soc. Mass. Spectrom. 2021;32:1964–1975. doi: 10.1021/jasms.0c00425. PubMed DOI

Bollinger JG, et al. Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization. Anal. Chem. 2010;82:6790–6796. doi: 10.1021/ac100720p. PubMed DOI PMC

Pham HT, Ly T, Trevitt AJ, Mitchell TW, Blanksby SJ. Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry. Anal. Chem. 2012;84:7525–7532. doi: 10.1021/ac301652a. PubMed DOI

Astudillo AM, et al. Occurrence and biological activity of palmitoleic acid isomers in phagocytic cells. J. Lipid Res. 2018;59:237–249. doi: 10.1194/jlr.M079145. PubMed DOI PMC

Lawrence P, Brenna JT. Acetonitrile covalent adduct chemical ionization mass spectrometry for double bond localization in non-methylene-interrupted polyene fatty acid methyl esters. Anal. Chem. 2006;78:1312–1317. doi: 10.1021/ac0516584. PubMed DOI

Wang DH, et al. Unusual polymethylene-interrupted, Δ5 monounsaturated and omega-3 fatty acids in sea urchin (Arbacia punctulata) from the Gulf of Mexico identified by solvent mediated covalent adduct chemical ionization mass spectrometry. Food Chem. 2022;371:131131. doi: 10.1016/j.foodchem.2021.131131. PubMed DOI

Zhang W, et al. Online photochemical derivatization enables comprehensive mass spectrometric analysis of unsaturated phospholipid isomers. Nat. Commun. 2019;10:79. doi: 10.1038/s41467-018-07963-8. PubMed DOI PMC

Zhao J, Fang M, Xia Y. A liquid chromatography-mass spectrometry workflow for in-depth quantitation of fatty acid double bond location isomers. J. Lipid Res. 2021;62:100110. doi: 10.1016/j.jlr.2021.100110. PubMed DOI PMC

Vrkoslav V, Háková M, Pecková K, Urbanová K, Cvačka J. Localization of double bonds in wax esters by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry utilizing the fragmentation of acetonitrile-related adducts. Anal. Chem. 2011;83:2978–2986. doi: 10.1021/ac1030682. PubMed DOI

Horká P, Vrkoslav V, Kindl J, Schwarzová-Pecková K, Cvačka J. Structural characterization of unusual fatty acid methyl esters with double and triple bonds using HPLC/APCI-MS2 with acetonitrile in-source derivatization. Molecules. 2021;26:6468. doi: 10.3390/molecules26216468. PubMed DOI PMC

Macias LA, Garza KY, Feider CL, Eberlin LS, Brodbelt JS. Relative quantitation of unsaturated phosphatidylcholines using 193 nm ultraviolet photodissociation parallel reaction monitoring mass spectrometry. J. Am. Chem. Soc. 2021;143:14622–14634. doi: 10.1021/jacs.1c05295. PubMed DOI PMC

Thomas MC, Mitchell TW, Blanksby SJ. Ozonolysis of phospholipid double bonds during electrospray ionization:  a new tool for structure determination. J. Am. Chem. Soc. 2006;128:58–59. doi: 10.1021/ja056797h. PubMed DOI

Thomas MC, et al. Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions. Anal. Chem. 2008;80:303–311. doi: 10.1021/ac7017684. PubMed DOI

Poad BLJ, et al. Ozone-induced dissociation on a modified tandem linear ion-trap: Observations of different reactivity for isomeric lipids. J. Am. Soc. Mass. Spectrom. 2010;21:1989–1999. doi: 10.1016/j.jasms.2010.08.011. PubMed DOI

Poad BLJ, et al. High-pressure ozone-induced dissociation for lipid structure elucidation on fast chromatographic timescales. Anal. Chem. 2017;89:4223–4229. doi: 10.1021/acs.analchem.7b00268. PubMed DOI

Marshall DL, et al. Mapping unsaturation in human plasma lipids by data-independent ozone-induced dissociation. J. Am. Soc. Mass. Spectrom. 2019;30:1621–1630. doi: 10.1007/s13361-019-02261-z. PubMed DOI

Claes BSR, et al. Mass spectrometry imaging of lipids with isomer resolution using high-pressure ozone-induced dissociation. Anal. Chem. 2021;93:9826–9834. doi: 10.1021/acs.analchem.1c01377. PubMed DOI PMC

Liu X, et al. Development of a miniature mass spectrometry system for point-of-care analysis of lipid isomers based on ozone-induced dissociation. Anal. Chem. 2022;94:13944–13950. doi: 10.1021/acs.analchem.2c03112. PubMed DOI

Young RSE, et al. Apocryphal FADS2 activity promotes fatty acid diversification in cancer. Cell Rep. 2021;34:108738. doi: 10.1016/j.celrep.2021.108738. PubMed DOI

Young RSE, et al. Identification of carbon-carbon double bond stereochemistry in unsaturated fatty acids by charge-remote fragmentation of fixed-charge derivatives. Anal. Chem. 2022;94:16180–16188. doi: 10.1021/acs.analchem.2c03625. PubMed DOI

Kind T, et al. LipidBlast in silico tandem mass spectrometry database for lipid identification. Nat. Methods. 2013;10:755–758. doi: 10.1038/nmeth.2551. PubMed DOI PMC

Tsugawa H, et al. A lipidome atlas in MS-DIAL 4. Nat. Biotechnol. 2020;38:1159–1163. doi: 10.1038/s41587-020-0531-2. PubMed DOI

Wolrab D, et al. LipidQuant 1.0: automated data processing in lipid class separation–mass spectrometry quantitative workflows. Bioinformatics. 2021;37:4591–4592. doi: 10.1093/bioinformatics/btab644. PubMed DOI

Husen P, et al. Analysis of lipid experiments (ALEX): a software framework for analysis of high-resolution shotgun lipidomics data. PLoS ONE. 2013;8:e79736. doi: 10.1371/journal.pone.0079736. PubMed DOI PMC

Pauling JK, et al. Proposal for a common nomenclature for fragment ions in mass spectra of lipids. PLoS ONE. 2017;12:e0188394. doi: 10.1371/journal.pone.0188394. PubMed DOI PMC

Herzog R, et al. A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language. Genome Biol. 2011;12:R8. doi: 10.1186/gb-2011-12-1-r8. PubMed DOI PMC

Herzog R, Schwudke D, Shevchenko A. LipidXplorer: software for quantitative shotgun lipidomics compatible with multiple mass spectrometry platforms. Curr. Protoc. Bioinform. 2013;43:14.12. 11–14.12. 30. doi: 10.1002/0471250953.bi1412s43. PubMed DOI

Hellhake S, et al. Non-targeted and targeted analysis of oxylipins in combination with charge-switch derivatization by ion mobility high-resolution mass spectrometry. Anal. Bioanal. Chem. 2020;412:5743–5757. doi: 10.1007/s00216-020-02795-2. PubMed DOI PMC

Chen L, et al. Metabolite discovery through global annotation of untargeted metabolomics data. Nat. Methods. 2021;18:1377–1385. doi: 10.1038/s41592-021-01303-3. PubMed DOI PMC

Koelmel JP, Ulmer CZ, Jones CM, Yost RA, Bowden JA. Common cases of improper lipid annotation using high-resolution tandem mass spectrometry data and corresponding limitations in biological interpretation. Biochim Biophys. Acta Mol. Cell Biol. Lipids. 2017;1862:766–770. doi: 10.1016/j.bbalip.2017.02.016. PubMed DOI PMC

Kirkwood KI, et al. Utilizing Skyline to analyze lipidomics data containing liquid chromatography, ion mobility spectrometry and mass spectrometry dimensions. Nat. Protoc. 2022;17:2415–2430. doi: 10.1038/s41596-022-00714-6. PubMed DOI PMC

MacLean B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26:966–968. doi: 10.1093/bioinformatics/btq054. PubMed DOI PMC

Pino LK, et al. The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics. Mass Spectrom. Rev. 2020;39:229–244. doi: 10.1002/mas.21540. PubMed DOI PMC

Adams KJ, et al. Skyline for small molecules: a unifying software package for quantitative metabolomics. J. Proteome Res. 2020;19:1447–1458. doi: 10.1021/acs.jproteome.9b00640. PubMed DOI PMC

Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid Res. 2008;49:1137–1146. doi: 10.1194/jlr.D700041-JLR200. PubMed DOI PMC

Quehenberger O, Dennis EA. The human plasma lipidome. N. Engl. J. Med. 2011;365:1812–1823. doi: 10.1056/NEJMra1104901. PubMed DOI PMC

Phinney KW, et al. Development of a standard reference material for metabolomics research. Anal. Chem. 2013;85:11732–11738. doi: 10.1021/ac402689t. PubMed DOI PMC

Micalizzi G, et al. Rapid and miniaturized qualitative and quantitative gas chromatography profiling of human blood total fatty acids. Anal. Bioanal. Chem. 2020;412:2327–2337. doi: 10.1007/s00216-020-02424-y. PubMed DOI

Benner Jr, B. A. & Murray, J. A. NIST Fatty Acid Quality Assurance Program 2017 Final Report, (National Institute of Standards and Technology, Gaithersburg, MD, 2019).

Schantz, M. M., Powers, C. D. & Schleicher, R. L. Interlaboratory Analytical Comparison Study of Total Fatty Acid Concentrations in Human Serum: Results for Exercise 01: QA12FASER01 (US Department of Commerce, National Institute of Standards and Technology, 2013).

White JB, et al. Equivalent carbon number and interclass retention time conversion enhance lipid identification in untargeted clinical lipidomics. Anal. Chem. 2022;94:3476–3484. doi: 10.1021/acs.analchem.1c03770. PubMed DOI

Phillips GB, Dodge JT. Composition of phospholipids and of phospholipid fatty acids of human plasma. J. Lipid Res. 1967;8:676–681. doi: 10.1016/S0022-2275(20)38891-X. PubMed DOI

Christinat N, Morin-Rivron D, Masoodi M. High-throughput quantitative lipidomics analysis of nonesterified fatty acids in human plasma. J. Proteome Res. 2016;15:2228–2235. doi: 10.1021/acs.jproteome.6b00198. PubMed DOI

Pédrono F, et al. Sciadonic acid derived from pine nuts as a food component to reduce plasma triglycerides by inhibiting the rat hepatic Δ9-desaturase. Sci. Rep. 2020;10:6223. doi: 10.1038/s41598-020-63301-3. PubMed DOI PMC

Shibahara A, Yamamoto K, Shinkai K, Nakayama T, Kajimoto G. cis-9,cis-15-Octadecadienoic acid: a novel fatty acid found in higher plants. Biochim. Biophys. Acta Lipids Lipid Metab. 1993;1170:245–252. doi: 10.1016/0005-2760(93)90006-U. PubMed DOI

Paradis M, Ackman RG. Potential for employing the distribution of anomalous non-methylene-interrupted dienoic fatty acids in several marine invertebrates as part of food web studies. Lipids. 1977;12:170–176. doi: 10.1007/BF02533289. PubMed DOI

Wang DH, Wang Z, Chen R, Brenna JT. Characterization and semiquantitative analysis of novel ultratrace C10–24 monounsaturated fatty acid in bovine milkfat by solvent-mediated covalent adduct chemical ionization (CACI) MS/MS. J. Agric. Food Chem. 2020;68:7482–7489. doi: 10.1021/acs.jafc.0c03031. PubMed DOI

Murawski U, Egge H, György P, Zilliken F. Identification of non-methylene-interrupted cis, cis-octadecadienoic acids in human milk. FEBS Lett. 1971;18:290–292. doi: 10.1016/0014-5793(71)80468-4. PubMed DOI

Hauff S, Vetter W. Exploring the fatty acids of vernix caseosa in form of their methyl esters by off-line coupling of non-aqueous reversed phase high performance liquid chromatography and gas chromatography coupled to mass spectrometry. J. Chromatogr. A. 2010;1217:8270–8278. doi: 10.1016/j.chroma.2010.10.088. PubMed DOI

Míková R, et al. Newborn boys and girls differ in the lipid composition of vernix caseosa. PLoS ONE. 2014;9:e99173. doi: 10.1371/journal.pone.0099173. PubMed DOI PMC

Narreddula VR, et al. Introduction of a fixed-charge, photolabile derivative for enhanced structural elucidation of fatty acids. Anal. Chem. 2019;91:9901–9909. doi: 10.1021/acs.analchem.9b01566. PubMed DOI

Takigawa H, Nakagawa H, Kuzukawa M, Mori H, Imokawa G. Deficient production of hexadecenoic acid in the skin is associated in part with the vulnerability of atopic dermatitis patients to colonization by Staphylococcus aureus. Dermatology. 2005;211:240–248. doi: 10.1159/000087018. PubMed DOI

Funkhouser LJ, Bordenstein SR. Mom knows best: the universality of maternal microbial transmission. PLoS Biol. 2013;11:e1001631. doi: 10.1371/journal.pbio.1001631. PubMed DOI PMC

Wolrab D, et al. Lipidomic profiling of human serum enables detection of pancreatic cancer. Nat. Commun. 2022;13:124. doi: 10.1038/s41467-021-27765-9. PubMed DOI PMC

Pascual G, et al. Targeting metastasis-initiating cells through the fatty acid receptor CD36. Nature. 2017;541:41–45. doi: 10.1038/nature20791. PubMed DOI

Küçüksayan, E. et al. Sapienic Acid Metabolism Influences Membrane Plasticity and Protein Signaling in Breast Cancer Cell Lines. Cells11, 225 (2022). PubMed PMC

Hoy AJ, Nagarajan SR, Butler LM. Tumour fatty acid metabolism in the context of therapy resistance and obesity. Nat. Rev. Cancer. 2021;21:753–766. doi: 10.1038/s41568-021-00388-4. PubMed DOI

Cao W, et al. Large-scale lipid analysis with C=C location and sn-position isomer resolving power. Nat. Commun. 2020;11:375. doi: 10.1038/s41467-019-14180-4. PubMed DOI PMC

Kirschbaum C, et al. Establishing carbon–carbon double bond position and configuration in unsaturated fatty acids by gas-phase infrared spectroscopy. Chem. Sci. 2023;14:2518–2527. doi: 10.1039/D2SC06487A. PubMed DOI PMC

Vriens K, et al. Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity. Nature. 2019;566:403–406. doi: 10.1038/s41586-019-0904-1. PubMed DOI PMC

Else PL. The highly unnatural fatty acid profile of cells in culture. Prog. Lipid Res. 2020;77:101017. doi: 10.1016/j.plipres.2019.101017. PubMed DOI

Guillou H, Zadravec D, Martin PGP, Jacobsson A. The key roles of elongases and desaturases in mammalian fatty acid metabolism: Insights from transgenic mice. Prog. Lipid Res. 2010;49:186–199. doi: 10.1016/j.plipres.2009.12.002. PubMed DOI

Brenna JT, Kothapalli KSD. New understandings of the pathway of long-chain polyunsaturated fatty acid biosynthesis. Curr. Opin. Clin. Nutr. Metab. Care. 2022;25:60–66. doi: 10.1097/MCO.0000000000000810. PubMed DOI

Ellis SR, et al. Automated, parallel mass spectrometry imaging and structural identification of lipids. Nat. Methods. 2018;15:515–518. doi: 10.1038/s41592-018-0010-6. PubMed DOI

Scott JS, Nassar ZD, Swinnen JV, Butler LM. Monounsaturated fatty acids: key regulators of cell viability and intracellular signaling in cancer. Mol. Cancer Res. 2022;20:1354–1364. doi: 10.1158/1541-7786.MCR-21-1069. PubMed DOI

Hamilton BR, et al. Mapping enzyme activity on tissue by functional mass spectrometry imaging. Angew. Chem. Int. Ed. 2020;59:3855–3858. doi: 10.1002/anie.201911390. PubMed DOI PMC

Randolph CE, Blanksby SJ, McLuckey SA. Enhancing detection and characterization of lipids using charge manipulation in electrospray ionization-tandem mass spectrometry. Chem. Phys. Lipids. 2020;232:104970. doi: 10.1016/j.chemphyslip.2020.104970. PubMed DOI PMC

Wang M, Han RH, Han X. Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach. Anal. Chem. 2013;85:9312–9320. doi: 10.1021/ac402078p. PubMed DOI PMC

Yang K, Dilthey BG, Gross RW. Identification and quantitation of fatty acid double bond positional isomers: a shotgun lipidomics approach using charge-switch derivatization. Anal. Chem. 2013;85:9742–9750. doi: 10.1021/ac402104u. PubMed DOI PMC

Menzel, J. P. Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome. jphmenzel/jpmlipidomics. 10.5281/zenodo.7930133 (2023). PubMed PMC

Menzel, J. P. et al. Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome. QUT Research Data Finder, 10.25912/RDF_1667992514317 (2022). PubMed PMC

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