Fatty acids (FA) play vital biological roles as energy sources, signaling molecules and key building blocks of complex lipids in cell membranes. Modifications to FA structure and composition are associated with the onset and progression of a number of chronic diseases. Consequently, the sensitive detection and unambiguous structure elucidation of FA is integral to the advancement of biomedical sciences. Recent advances in FA analysis have taken advantage of wet chemical derivatization to enhance detection and drive unique fragmentation in tandem mass spectrometry protocols. Here, we significantly further this approach through demonstrating gas-phase charge inversion of singly deprotonated FA ions, [M − H]−, using doubly charged tris-phenanthroline alkaline earth metal complexes, [Cat(Phen)3]2+ (Cat = Mg2+, Ca2+, Sr2+, or Ba2+). Metal cationized FA, [M − H + Cat]+ are obtained after the gas-phase ion/ion reaction. Low-energy collision-induced dissociation (CID) of the [M − H + Cat]+ cations facilitates double bond localization for a variety of monounsaturated and polyunsaturated FAs. Ultimately, detailed characterization presented unambiguous distinction amongst FA double bond positional isomers, such as n-3 and n-6 isomers. The method was successfully used to identify the FA profile of corn oil, including double bond localization for unsaturated FAs present.

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.

Representing the most fundamental lipid class, fatty acids (FA) play vital biological roles serving as energy sources, cellular signaling molecules, and key architectural components of complex lipids. Direct infusion electrospray ionization spectrometry, also known as shotgun lipidomics, has emerged as a rapid and powerful toolbox for lipid analysis. While shotgun lipidomics can be a sensitive approach to FA detection, the diverse molecular structure of FA presents challenges for unambiguous identification and the relative quantification of isomeric contributors. In particular, pinpointing double bond position(s) in unsaturated FA and determining the relative contribution of double bond isomers has limited the application of the shotgun approach. Recently, we reported the use of gas-phase ion/ion reactions to facilitate the identification of FA. Briefly, singly deprotonated FA anions undergo charge inversion when reacted in the gas phase with trisphenanthroline magnesium dications by forming [FA-H + MgPhen] + complex ions. These charge-inverted FA complex cations fragment upon ion-trap collision-induced dissociation (CID) to generate product ion spectra unique to individual FA isomers. Herein, we report the development of a mass spectral library comprised of [FA-H + MgPhen] + product ion spectra. The developed FA library permits confident FA identification, including polyunsaturated FA isomers. Furthermore, we demonstrate the ability to determine relative contributions of isomeric FA using multiple linear regression analysis paired with gas-phase ion/ion reactions. We successfully applied the presented method to generate a FA profile for bovine liver phospholipidome based entirely on gas-phase chemistries.

Shotgun lipid analysis based on electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in lipidomic studies. One challenge for the shotgun approach is the discrimination of lipid isomers and isobars. Gas-phase charge inversion via ion/ion reactions has been used as an effective method to identify multiple isomeric/isobaric components in a single MS peak by exploiting the distinctive functionality of different lipid classes. In doing so, fatty acyl chain information can be obtained without recourse to condensed-phase separations or derivatization. This method alone, however, cannot provide carbon-carbon double bond (C=C) location information from fatty acyl chains. Herein, we provide an enhanced method pairing photochemical derivatization of C=C via the Paterno-Buchi reaction with charge inversion ion/ion tandem mass spectrometry. This method was able to provide gas-phase separation of phosphatidylcholines and phosphatidylethanolamines, the fatty acyl compositions, and the C=C location within each fatty acyl chain. We have successfully applied this method to bovine liver lipid extracts and identified 40 molecular species of glycerophospholipids with detailed structural information including head group, fatty acyl composition, and C=C location.

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.