Abstract
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 MS(n) platform paired with ion/ion chemistry permitted identification of major, and some minor, isomeric contributors that are unresolved using conventional ESI-MS/MS.