Incorporation of the Paternò–Büchi reaction into mass spectrometry-based systems for lipid structural characterization
Creators: name: Franklin, Elissia T
Publisher: name: Purdue University Graduate School
Year: 2019
Subjects: Analytical chemistry not elsewhere classified

description: Lipids are important cellular biomolecules that perform essential functional and biological roles. For instance, lipids in the cell are the compartmentalizer for the cytoplasm and an energy storage unit. The knowledge surrounding lipids is abundant, yet there is still so much to uncover. There are many categories of lipids and within each category the structural composition is extremely diverse. In turn, the dramatic structural complexity of lipids demands analytical methods capable of providing in-depth structural characterization of individual molecular structures. However, lipid structural elucidation has remained challenging, namely due to the presence of isomeric and isobaric species with a complex mixture. In particular, isomeric/isobaric lipid structures arise from variations in class, headgroup, fatty acyl chain, <i>sn</i>-position, and/or carbon-carbon double bond (C=C) position(s). Recently, recent research suggests C=C composition impacts lipid physical properties, metabolic fate, and intermolecular interactions. Thus, analytical strategies capable of localizing sites of unsaturation are of interest in the lipidomics community. Mass spectrometry (MS) is a leading tool for lipid analysis. Electrospray ionization (ESI), a soft ionization method, is the most commonly used method for lipid ionization as a means of taking the ions from liquid-phase to gas-phase without extensive decomposition of the species. Utilizing ESI-MS, lipids can be identified at a sum compositional level via accurate mass measurements. . With tandem mass spectrometers, lipid ions can be further probed, utilizing tandem-MS (MS/MS) to generate structurally informative product ion spectra that facilitate the assignment of lipid molecular structure. More so, gas-phase ion/ion reactions represent a unique MS-based technique that has improved the analysis of lipids structures. Gas-phase ion/ion reactions allow for lipid species to be charge inverted from one polarity to the opposite polarity. This reaction enables lipids to be ionized in a polarity that is optimal for class identification and further investigated in the opposite polarity where more structural information is obtained. All the information provided is captured without the requirement of multiple solution conditions which is necessary when analyzing in both polarities. In the case of charge inverted lipids from positive ion mode to negative ion mode, fatty acyl composition can be obtained; however, C=C information is lacking. MS can also be paired with other analytically technologies to assist with lipid analysis. One of those technologies is liquid chromatography (LC), which allows for the separation of lipids based on different characteristic depending on the column type being used. Reverse-phase LC (RPLC) allows for the separation of lipid molecular species based on structural composition. RPLC-MS/MS benefits from the ability to separate lipids and determine their fatty acyl chain composition but it is difficult to specify C=C location with the use of a synthetic standard that is identical to each molecular species being analyzed. Commonality between the gas-phase ion/ion reactions for charge inversion of lipids and RPLC-MS/MS approaches is the inability to provide C=C coverage. In-solution and unique ion activation techniques have been developed for seeking such information. The Paternò–Büchi reaction is a UV-initiated [2 + 2]-cycloaddition of an excited carbonyl containing compound onto an olefin group. This reaction can be initiated onto the alkene group within an unsaturated lipid aliphatic chain to form an oxetane ring modification. There are two product ions that can be formed upon each unsaturation site due to a lack of regioselectivity the reagent can attach at either side of the C=C. The modified lipids can be taken into gas-phase and collisionally activated via low-energy collision induced dissociation, generating product ions indictive of C=C position(s). The work herein shows the incorporation of the PB reaction into the gas-phases ion/ion reaction and RPLC-MS/MS apparatuses for C=C localization. The methods have been applied to the lipid extracts of bovine liver and human plasma for confident molecule species determination.