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Localization of Carbon-Carbon Double Bond and Cyclopropane Sites in Cardiolipins via Gas-Phase Charge Inversion Reactions.

ABSTRACT: Cardiolipins (CLs) are comprised of two phosphatic acid moieties bound to a central glycerol backbone and are substituted with four acyl chains. Consequently, a vast number of distinct CL structures are possible in different biological contexts, representing a significant analytical challenge. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has become a widely used approach for the detection, characterization, and quantitation of complex lipids, including CLs. Central to this approach is fragmentation of the [CLs - H]^- or [CL - 2H]^2- anions by collision-induced dissociation (CID). Product ions in the resulting tandem mass spectra confirm the CL subclass assignment and reveal the numbers of carbons and degrees of unsaturation in each of the acyl chains. Conventional CID, however, affords limited structural elucidation of the fatty acyl chains, failing to discriminate isomers arising from different site(s) of unsaturation or cyclopropanation and potentially obscuring their metabolic origins. Here, we report the application of charge inversion ion/ion chemistry in the gas phase to enhance the structural elucidation of CLs. Briefly, CID of [CL - H]^2- anions generated via negative ion ESI allowed for the assignment of individual fatty acyl substituents and phosphatidic acid moieties. Next, gas-phase derivatization of the resulting CL product ions, including fatty acyl carboxylate anions, was effected with gas-phase ion/ion charge inversion reactions with tris-phenanthroline magnesium reagent dications. Subsequent isolation and activation of the charge-inverted fatty acyl complex cations permitted the localization of both carbon-carbon double bond and cyclopropane motifs within each of the four acyl chains of CLs. This approach was applied to the de novo elucidation of unknown CLs in a biological extract revealing distinct isomeric populations and regiochemical relationships between double bonds and carbocyles.

SUBMITTER: Randolph CE

PROVIDER: S-EPMC8557092 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Shotgun lipidomics has recently gained popularity for lipid analysis. Conventionally, shotgun analysis of glycerophospholipids via direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides glycerophospholipid (GPL) class (i.e., headgroup composition) and fatty acyl composition. Reliant on low-energy collision-induced dissociation (CID), traditional ESI-MS/MS fails to define fatty acyl regiochemistry along the glycerol backbone or carbon-carbon double bond position(s) in unsaturated fatty acyl substituents. Therefore, isomeric GPLs are often unresolved, representing a significant challenge for shotgun-MS approaches. We developed a top-down shotgun-MS method utilizing gas-phase ion/ion charge inversion chemistry that provides near-complete GPL structural identification. First, in negative ion mode, CID of mass-selected GPL anions generates fatty acyl carboxylate anions via fragmentation of ester bonds linking the fatty acyl substituents at the sn-1 and sn-2 positions of the glycerol backbone. Product anions, including fatty acyl carboxylate ions, were then derivatized in the mass spectrometer via an ion/ion charge inversion reaction with tris-phenanthroline magnesium dications. Subsequent CID of charge-inverted fatty acyl complex cations yielded isomer-specific product ion spectra that permit (i) unambiguous assignment of carbon-carbon double bond position(s) and (ii) relative quantitation of isomeric fatty acyl substituents. The outlined strategy was applied to the analysis of targeted GPLs extracted from human plasma, including several proposed plasma biomarkers. A single experiment thus facilitates assignment of the GPL headgroup, fatty acyl composition, carbon-carbon double bond position(s) in unsaturated fatty acyl chains, and, in some cases, fatty acyl sn-position and relative abundances for isomeric fatty acyl substituents. Ultimately, this MSn platform paired with ion/ion chemistry permitted identification of major, and some minor, isomeric contributors that are unresolved using conventional ESI-MS/MS.

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Localization of Carbon-Carbon Double Bond and Cyclopropane Sites in Cardiolipins via Gas-Phase Charge Inversion Reactions.

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