Giovanni Venturaa,b, Vito Nettisa, Liam McDonnellC, Noa NdimurwankoC,
Francesco GrecoC, Tommaso R.I. Cataldia,b Cosima D. Calvanoa,b

aDipartimento di Chimica, bCentro di Ricerca Interdipartimentale SMART, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126, Bari, Italy, 
cFondazione Pisana per la Scienza, Via Ferruccio Giovannini, 13, 56017 San Giuliano Terme PI, Pisa, Italy

The selective epoxidation of carbon–carbon double bonds with meta-chloroperbenzoic acid (m-CPBA) converts unsaturated analytes into epoxides in a single concerted step and enables double-bond localization by tandem mass spectrometry (MS/MS) through the generation of diagnostic product ions that report C=C positions [1-4]. When m-CPBA derivatization is implemented on instruments without MS3 capability, such as certain high-resolution Orbitrap platforms, unambiguous assignment in intact phospholipids bearing unsaturations on both fatty-acyl chains (FACs) can be challenging because conventional MS/MS produces composite spectra derived from both chains [4].

Here we present a liquid chromatography–high-resolution mass spectrometry (LC–HRMS) workflow in which source-induced dissociation (SID) is applied prior to precursor isolation to liberate FACs from intact phospholipids [5]. The resulting epoxidated free fatty acids signals are then individually isolated and subjected to MS/MS, yielding clear chain-specific fragmentation patterns that enable confident double-bond localization without the need for MS3 acquisition..

We demonstrate the utility of this integrated strategy by analyzing pancreatic ductal adenocarcinoma (PDAC)-derived cells via a combination of Hydrophilic interaction liquid chromatography (HILIC) and reversed phase approach, where it provides robust double-bond mapping across a complex lipid background. In addition, we developed an HILIC-ESI(-)-all ion fragmentation (AIF) approach to understand the occurrence of different FACs isomers in each lipid class

Acknowledgements: this work is supported by project POS4 Life Science and by “LIpidome of extracellular VEscicles in hypoxic pancreatic cancer” acronimo “LIVE” CUP - H93C24000620006, Bando a cascata emanato dallo Spoke 6 del Tuscany Health Ecosystem a valere sulle risorse del PNRR, M4C2 - Investimento 1.5 – creazione e rafforzamento di “Ecosistemi dell’innovazione per la sostenibilità”, finanziato dall’Unione Europea – NEXTGENERATIONEU 

Keywords: Epoxidation,  pancreatic ductal adenocarcinoma, LC-ESI-FTMS

References

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