Comprehensive analysis of oxidized arachidonoyl-containing glycerophosphocholines using ion mobility spectrometry-mass spectrometry

The biological significance of oxidized arachidonoyl-containing glycerophosphocholines, exemplified by the oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), in pathological processes is well-established. However, despite their widespread use in redox lipidomics research, the precise chemical composition of the heterogeneous mixtures of oxPAPC generated in vitro -including the high prevalence of isomers and the oxidation mechanisms involved- remain inadequately understood. To address these knowledge gaps, we developed a multidimensional in-house database from a commercial oxPAPC preparation -employing Liquid Chromatography coupled to Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) and Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS). This database includes lipid names, retention times, accurate mass values (m/z), adduct profiles, MS/MS information, as well as collision cross-section (CCS) values. Our investigation elucidated 34 compounds belonging to distinct subsets of oxPAPC products, encompassing truncated, full-length, and cyclized variants. The integration of IMS-MS crucially facilitated: (i) structural insights among regioisomers, exemplified by the 5,6-PEIPC and 11,12-PEIPC epoxy-isoprostane derivatives, (ii) novel Collision Cross Section (CCS) values, and (iii) cleaner MS/MS spectra for elucidating the fragmentation mechanisms involved to yield specific fragment ions. These diagnostic ions were employed to successfully characterize full-length isomers present in human plasma samples from patients with mucormycosis. This comprehensive oxPAPC characterization not only advances the understanding of lipid peroxidation products but also enhances analytical capabilities for in vitro-generated oxidized mixtures. The implementation of this robust database, containing multiple orthogonal (i.e., independent) pieces of information, will serve as a comprehensive resource for the field.