Double bond localization in unsaturated rhamnolipid precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids by liquid chromatography–mass spectrometry applying online Paternò–Büchi reaction

Abstract

Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two L-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ-Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.

Multifunctional Reactive MALDI Matrix Enabling High-Lateral Resolution Dual Polarity MS Imaging and Lipid C═C Position-Resolved MS2 Imaging

“…Because BzPy attachment was observed for unsaturated and saturated lipids, BzPy most likely also reacts with the head group, in a reaction other than PB functionalization, or attaches to lipids in a noncovalent manner. This is also consistent with the reports by Xia31 and Hayen30 about PB reaction by-products in in-solution reactions. The numbers of lipids with or without BzPy attachment are compared in Figure3.…”

“…24−28 The latter functionalization scheme, pioneered by Ma and Xia, 29 has been used by numerous groups to assign DB positions in direct infusion and LC−MS workflows. 30,31 Lipid DB isomer assignment from surfaces or local alterations of lipid isomer abundances in tissues, however, require desorption-based ionization methods. 14,20,23,32 33,34 In one of our recent studies, we identified benzophenone (BPh) as a compound that can act as a MALDI matrix and reacts with lipids in a PB reaction during laser irradiation, a process termed PB-reactive MALDI.…”

Advanced tandem mass spectrometry in metabolomics and lipidomics—methods and applications

“…However, the discovery by Ma and Xia [114] that light-induced Paternò-Büchi (PB) reactions can aid DB position assignments with relative ease and with inexpensive equipment has sparked the interest in chemical derivatization strategies that enable structure elucidation. In particular, most recent derivatization strategies target lipid DBs or adjacent carbon atoms and are based on PB [114][115][116][117][118][119][120][121][122], epoxidation [123][124][125], ozonolysis [126], or hydroxylation reactions [127] (Scheme 2).…”

“…PB reactions with acpy and other carbonyl compounds have been used not only to analyze standards but also to investigate complex lipid extracts from body fluids, cells, or tissues revealing DB positions and sometimes sn-isomers for CEs [116,130], FAs [116], GPs [121], and SLs [131]. Recently, PB methods have been extended to also target small FA metabolites [117][118][119] or have been adapted to benefit other advanced tandem MS tools such as UVPD [115] or ion/ion reactions [122].…”

Multifunctional Reactive MALDI Matrix Enabling High-Lateral Resolution Dual Polarity MSI and Lipid C=C Position-Resolved MS2I

“…[24][25][26][27][28] The latter functionalization scheme, pioneered by Ma and Xia, 29 has been used by numerous groups to assign DB positions in direct infusion and LC-MS workflows. 30,31 Lipid DB isomer assignment from surfaces or local alterations of lipid isomer abundances in tissue, however, require desorption-based ionization methods. 14,23,20,32 For example, MALDI MS and MS imaging (MSI) tools were developed that enable pinpointing lipid DB positions.…”