Rationale: The emergence of new mass spectrometry (MS) dissociation methods has highlighted lipid isomers as new biomarkers. Only a few commercial methods without derivatization are available to characterize phosphatidylcholines (PCs) at the isomeric level. We propose to use electron transfer dissociation (ETD) as a method to determine the position of both double bonds and stereo numbering (sn) on glycerol for PC species.
Methods: Doubly charged PCs were analyzed using alkali salts to promote the formation of [PC + 2Alk]2+ species. ETD-MS2 experiments were performed using a quadrupole ion trap mass spectrometer with an ESI source to annotate sn-positions. ETD-CID-MS3 experiments were then done on ETnoD [M + 2Alk]+• or [M + 2Alk-N(CH3)3]+• species to localize double bond positions. Density functional theory (DFT) calculations and cyclic ion mobility spectrometry c-IMS-MS/MS experiments were used to support fragmentation assumptions.
Results: ETD-MS2 experiments exhibit a systematic sn-2 favorable cleavage, allowing sn-positioning through a radical cation-driven mechanism supported by DFT calculations. This behavior contrasts with CID experiments, in which the initial positioning of alkali cations influences ester bond cleavage, as shown by c-IMS-MS/MS experiments. The dissociation process studied for ETD-CID-MS3 experiments on 10 different PC isomers allowed us to localize double bonds for either monounsaturated or polyunsaturated species.
Conclusions: The dissociation rules established for ETD-MS2 and ETD-CID-MS3 experiments on PC isomers enable their annotation at the isomeric level without instrumental modification or complex sample preparation. This method could be easily coupled to LC separation using post-column introduction of an alkali salt for complex mixture analysis.
Keywords: double bond location; electron transfer dissociation; isomer characterization; phosphatidylcholine.
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