Gas-Phase Ion/Ion Reactions to Enable Radical-Directed Dissociation of Fatty Acid Ions: Application to Localization of Methyl Branching.

Methyl branching on the carbon chains of fatty acids and fatty esters is among the structural variations encountered with fatty acids and fatty esters. Branching in fatty acid/ester chains is particularly prominent in bacterial species and, for example, in vernix caseosa and sebum. The distinction of branched chains from isomeric straight-chain species and the localization of branching can be challenging to determine by mass spectrometry (MS). Condensed-phase derivatization strategies, often used in conjunction with separations, are most commonly used to address the identification and characterization of branched fatty acids. In this work, a gas-phase ion/ion strategy is presented that obviates condensed-phase derivatization and introduces a radical site into fatty acid ions to facilitate radical-directed dissociation (RDD). The gas-phase approach is also directly amenable to fatty acid anions generated via collision-induced dissociation from lipid classes that contain fatty esters. Specifically, divalent magnesium complexes bound to two terpyridine ligands that each incorporate a ((2,2,6,6-tetramethyl-1-piperidine-1-yl)oxy) (TEMPO) moiety are used to charge-invert fatty acid anions. Following the facile loss of one of the ligands and the TEMPO group of the remaining ligand, a radical site is introduced into the complex. Subsequent collision-induced dissociation (CID) of the complex exhibits preferred cleavages that localize the site(s) of branching. The approach is illustrated with iso-, anteiso-, and isoprenoid branched-chain fatty acids and an intact glycerophospholipid and is applied to a mixture of branched- and straight-chain fatty acids derived from Bacillus subtilis.

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry.

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon-carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP - H]- , generates FA anions, [FA - H]- . The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)3 ]2+ , to form charge inverted complex cations of the form [FA - H + MgPhen2 ]+ . CID of the complex generates product ion spectral patterns that allow for the identification of carbon-carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.

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

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.

Structural Elucidation of Ether Glycerophospholipids Using Gas-Phase Ion/Ion Charge Inversion Chemistry.

Ether lipids represent a unique subclass of glycerophospholipid (GPL) that possesses a 1-O-alkyl (i.e., plasmanyl subclass) or a 1-O-alk-1'-enyl (i.e., plasmenyl subclass) group linked at the sn-1 position of the glycerol backbone. As changes in ether GPL composition and abundance are associated with numerous human pathologies, analytical strategies capable of providing high-level structural detail are desirable. While mass spectrometry (MS) has emerged as a prominent tool for lipid structural elucidation in biological extracts, distinctions between the various isomeric forms of ether-linked GPLs have remained a significant challenge for tandem MS, principally due to similarities in the conventional tandem mass spectra obtained from the two ether-linked subclasses. To distinguish plasmanyl and plasmenyl GPLs, a multistage (i.e., MSn where n = 3 or 4) mass spectrometric approach reliant on low-energy collision-induced dissociation (CID) is required. While this method facilitates assignment of the sn-1 bond type (i.e., 1-O-alkyl versus 1-O-alk-1'-enyl), a composite distribution of isomers is left unresolved, as carbon-carbon double-bond (C=C) positions cannot be localized in the sn-2 fatty acyl substituent. In this study, we combine a systematic MSn approach with two unique gas-phase charge inversion ion/ion chemistries to elucidate ether GPL structures with high-level detail. Ultimately, we assign both the sn-1 bond type and sites of unsaturation in the sn-2 fatty acyl substituent using an entirely gas-phase MS-based workflow. Application of this workflow to human blood plasma extract permitted isomeric resolution and in-depth structural identification of major and, in some cases, minor isomeric contributors to ether GPLs that have been previously unresolved when examined via conventional methods.