Identification of Monomethyl Branched-Chain Lipids by a Combination of Liquid Chromatography Tandem Mass Spectrometry and Charge-Switching Chemistries

Abstract

While various mass spectrometric approaches have been applied to lipid analysis, unraveling the extensive structural diversity of lipids remains a significant challenge. Notably, these approaches often fail to differentiate between isomeric lipids�a challenge that is particularly acute for branched-chain fatty acids (FAs) that often share similar (or identical) mass spectra to their straight-chain isomers. Here, we utilize charge-switching strategies that combine ligated magnesium dications with deprotonated fatty acid anions. Subsequent activation of these charge inverted anions yields mass spectra that differentiate anteiso-branched-from straightchain and iso-branched-chain FA isomers with the predictable fragmentation enabling de novo assignment of anteiso branch points. The application of these charge-inversion chemistries in both gasand solution-phase modalities is demonstrated to assign the position of anteiso-methyl branch-points in FAs and, with the aid of liquid chromatography, can be extended to de novo assignment of additional branching sites via predictable fragmentation patterns as methyl branching site(s) move closer to the carboxyl carbon. The gas-phase approach is shown to be compatible with top-down structure elucidation of complex lipids such as phosphatidylcholines, while the integration of solution-phase charge-inversion with reversed phase liquid chromatography enables separation and unambiguous identification of FA structures within isomeric mixtures. Taken together, the presented charge-switching MS-based technique, in combination with liquid chromatography, enables the structural identification of branched-chain FA without the requirement of authentic methyl-branched FA reference standards.

The chemistry of the s- and p-block elements with 2,2′:6′,2′′-terpyridine ligands

“…Very recently, this approach was successfully applied to other substrates regarding structure elucidation and differentiation/separation of isomers: Phosphoinositides (essential signaling phospholipids in mammalian cellular membranes), 135 cerebrosides (a sphingolipid subclass), 136 and branched-chain fatty acids. 137 It is well-known that materials, which feature a high degreeof-anisotropy, either on the molecular or crystallographic level, possess a range of remarkable properties: Piezo-or ferroelectricity, non-linear optical behavior, birefringence, etc. The latter arises, if a crystal's refractive index in two perpendicular directions is unequal to zero.…”

“…A variety of biomolecules could be characterized and even the separation of isomers in the collision chamber could be achieved. [135][136][137] This straightforward technique will certainly be applied further to other classes of molecules. Regarding the heavier homologues of Mg, the formation of coordination polymers with notable birefringence has to be highlighted.…”

Formation and Tandem Mass Spectrometry of Doubly Charged Lipid-Metal Ion Complexes

“…43 Gas-phase charge inversion reactions performed in the McLuckey group enable the identification of C�C bond positions and cyclopropanation sites in cardiolipins 50 as well as methyl branching sites. 51 But also attachment of metal ions to GPLs resulting in singly 52 and doubly 53,54 charged lipid-metal ion complexes can be used to identify sn-isomers of PCs. To date, the majority of studies on structural lipidomics are performed with the goal of determining sn-isomers and C�C bond positions.…”

“…Another approach that enables CC position and sn -isomer assignment is OzID, pioneered by Blanksby et al Multiple groups have demonstrated the capabilities of PB functionalization as a means to determine CC bond positions in shotgun, coupled to LC and MS-imaging approaches. − The Xia group, for example, developed an MS 4 approach to determine the sn -isomeric composition as well as the CC double bond position for PCs . Gas-phase charge inversion reactions performed in the McLuckey group enable the identification of CC bond positions and cyclopropanation sites in cardiolipins as well as methyl branching sites . But also attachment of metal ions to GPLs resulting in singly and doubly , charged lipid-metal ion complexes can be used to identify sn -isomers of PCs.…”

Charge Inversion of Mono- and Dianions to Cations via Triply Charged Metal Complexes: Application to Lipid Mixtures

“…We have previously reported using divalent alkaline earth metals complexed with tris 1,10-phenanthroline (phen) − and 2,2′:6′,2″-terpyridine (terpy) − to charge-invert singly charged lipid species that allowed for in-depth structural characterization and isomeric differentiation. However, larger lipids such as Lipid A, cardiolipin, and phosphatidylinositol phosphate , preferentially ionize as dianions, precluding divalent metal complexes as suitable reagents for ion/ion reactions due to inevitable neutralization.…”