Introduction of a Fixed-Charge, Photolabile Derivative for Enhanced Structural Elucidation of Fatty Acids.

Fatty acids are a structurally diverse category of lipids with a myriad of biochemical functions, which includes their role as building blocks of more complex lipids (e.g., glycerophospholipids and triacylglycerols). Increasingly, the analysis of fatty acids is undertaken using liquid chromatography-mass spectrometry (LC-MS), due to its versatility in the detection of lipids across a wide range of concentrations and diversity of molecular structures and masses. Previous work has shown that fixed-charge pyridinium derivatives are effective in enhancing the detection of fatty acids in LC-MS workflows. Herein, we describe the development of two novel pyridinium fixed-charged derivatization reagents that incorporate a photolabile aryl iodide that is selectively activated by laser irradiation inside the mass spectrometer. Photodissociation mass spectra of fatty acids conjugated to 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) and 1-(4-(aminomethyl)-3-iodophenyl)pyridin-1-ium (3-I-AMPP+) derivatives reveal structurally diagnostic product ions. These spectra feature radical-directed dissociation of the carbon-carbon bonds within the fatty acyl chain, enabling structural assignments of fatty acids and discrimination of isomers that differ in site(s) of unsaturation, methyl branching or cyclopropanation. These derivatives are shown to be suitable for hyphenated LC-MS methods, and their predictable photodissociation behavior allows de novo identification of unusual fatty acids within a biological context.

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids.

The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.

Determination of ester position in isomeric (O-acyl)-hydroxy fatty acids by ion trap mass spectrometry.

RATIONALE: (O-acyl)-hydroxy fatty acids (OAHFAs) are a recently discovered class of endogenous lipids, generating significant interest for their correlation with enhanced glucose tolerance. Structural variants that differ in the position of the ester linkage have been described, including the ω-OAHFA sub-class, that plays a key role in stabilizing the human tear film. Developing analytical tools for rapid and unambiguous structural elucidation of OAHFAs is essential to understanding their diverse physiological functions. METHODS: Commercially available and synthesized OAHFA standards were dissolved in chloroform and subsequently diluted into methanol with 1.5 mM ammonium acetate. Negative ion collision-induced dissociation (CID) MSn spectra were acquired using chip-based nano-electrospray ionization (Advion TriVersa NanoMate) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). RESULTS: Major product ions observed during CID of [OAHFA - H]- ions readily identify the constituent fatty acid and hydroxy fatty acid; however, isomers are not easily distinguished. Interrogation of the hydroxy fatty acid and dehydrated hydroxy fatty acid product ions by MSn and ion-molecule reactions yielded diagnostic ions that readily pinpoint hydroxylation position and, thus, the OAHFA ester location. Conversely, these ions are characteristically absent in the MS3 spectra of ω-OAHFAs. Unimolecular dissociation mechanisms are proposed, which are shown to be consistent with prior isotopic labelling experiments. CONCLUSIONS: A mechanistic rationale is provided to explain the unimolecular dissociation of [OAHFA - H]- ions in an ion trap mass spectrometer, thus enabling near-complete de novo structural elucidation of OAHFAs in shotgun lipidomics workflows, even if synthetic standards are unavailable for comparison. Copyright © 2016 John Wiley & Sons, Ltd.