Imaging and Analysis of Isomeric Unsaturated Lipids through Online Photochemical Derivatization of Carbon–Carbon Double Bonds**

Abstract

Unraveling the complexity of the lipidome requires the development of novel approaches for the structural characterization of lipid species with isomer-level discrimination. Herein, we introduce an online photochemical approach for lipid isomer identification through selective derivatization of double bonds by reaction with singlet oxygen. Lipid hydroperoxide products are generated promptly after laser irradiation. Fragmentation of these species in a mass spectrometer produces diagnostic fragments revealing the C=C locations in the unreacted lipids. This approach uses an inexpensive light source and photosensitizer making it easy to incorporate into any lipidomics workflow. We demonstrate the utility of this approach for the shotgun profiling of C = C locations in different lipid classes present in tissue extracts using electrospray ionization (ESI) and ambient imaging of lipid species differing only by the location of C=C bonds using nanospray desorption electrospray ionization (nano-DESI).

Recent Advances of Ambient Mass Spectrometry Imaging and Its Applications in Lipid and Metabolite Analysis

“…After only 10 min of accelerated oxidation, the unsaturated lipid isomers in nude lung cancer tissue and human thyroid cancer tissues were successfully imaged by AFADESI. In 2021, for lipid isomer identification, Unsihuay et al reported an online photochemical derivatization approach, i.e., 1 O 2 reacted with lipids to form lipid hydroperoxides (LOOHs), which was then followed by collision-induced dissociation (CID) to obtain diagnostic peaks (Figure 6H) [106]. The Paternò-Büchi (PB) reaction is another commonly used method for identifying the location of C=C double bonds in unsaturated lipids and simplified PB reaction conditions were introduced by spraying the reagent solution on the tissue surface with only 10 min preprocessing [107].…”

“…(H) Mechanism experiment setup of the online single oxygen reaction with lipids. Reprinted with permission from the authors of [106]. Copyright (2021) Wiley-VCH GmbH.…”

Review of Recent Advances in Lipid Analysis of Biological Samples via Ambient Ionization Mass Spectrometry

“…In this workflow, the onset of the electro-epoxidation of the C=C bond could be controlled by tuning the voltage, and the diagnostic ions derived from the fragmentation of electro-epoxidized products by MS/MS were used to indicate the C=C positions. To circumvent the challenge in spatially resolving positional isomers in lipidomic analyses, the method for the online selective photochemical derivatization of the C=C bond by singlet oxygen ( 1 O 2 ) was proposed [2]. The mechanism of C=C bond oxidation by 1 O 2 (Figure 5B) can be briefly descripted as follows.…”

“…Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), as a hybrid soft ionization source based on IR-laser desorption followed by electrospray post-ionization, has been combined with on-tissue m-CPBA epoxidation for the in situ detection of FAs C=C positional isomers in rat liver and human bladder cancer tissue [65]. Furthermore, when coupling the 1 O 2 reaction with nano-DESI-MS, several isomeric lipids from mouse muscle and uterine tissues were directly detected [2]. Recently, an online coupling of the PB reaction and radical-direct fragmentation with in-capillary extraction nanoelectrospray ionization mass spectrometry (ICE-nanoESI-MS) [70] was developed in order to determine C=C locations and stereospecific numbering positions of potential lipid biomarkers for human colorectal cancer under an ambient condition; the results indicated that lysophospholipids could act as potential biomarkers related to the pathogenesis of human colorectal cancer.…”

“…Lipids are essential biomolecules involved in various cellular functions, including structural components, energy reservoirs, and signal molecules in biological systems [1,2]. Unsaturated lipids, as a subclass of lipids, structurally differ in the types of headgroups (such as phosphatidic acids (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS)), carbon-carbon double bond (C=C) locations, numbers, as well as cis/trans geometric isomerism, and the sn-position of the fatty acyl chain (Figure 1A), which complicate structural characterization [3,4].…”