Characterization of Oxidized Glycerophosphoethanolamines via Radical-Directed Dissociation Tandem Mass Spectrometry and the Paternò–Büchi Derivatization

Abstract

Oxidized glycerophosphoethanolamines (oxPEs) represent a subclass of bioactive lipids that have intricate roles in various physiological and pathological events. Conventional mass spectrometric methods cannot provide unambiguous information to locate the OH group and the sites of unsaturation. Herein, we report a combined strategy for in-depth structural characterization of oxPEs, including radical-directed dissociation tandem mass spectrometry (RDD-MS/MS) for localizing the OH group and the Paternò–Büchi derivatization coupled with tandem mass spectrometry for pinpointing carbon–carbon double-bond locations. The RDD-MS/MS method has been integrated on a reversed-phase liquid chromatography–mass spectrometry workflow. It enables the profiling of 24 distinct oxPE molecules with unequivocal assignment of the OH sites at nM sensitivity in bovine liver lipid extract treated by soybean 15-lipoxygenase. These findings showcase that the developed method has a good potential in analyzing biological systems where oxPEs may play important roles.

In-Depth Characterization of Sphingoid Bases via Radical-Directed Dissociation Tandem Mass Spectrometry

“…3-(2,2,6,6-Tetramethylpiperidin-1-yloxymethyl)-picolinic acid 2,5-dioxopyrrolidin-1-yl ester (TPN) was initially developed by the Gao group for free radical-initiated peptide sequencing (FRIPS) of hydrophobic peptides. , The relatively low BDE (30 kcal/mol) of the C–O bond facilitated the loss of 2,2,6,6-tetramethylpiperidine- N -oxyl radical (TEMPO·, 156 Da) upon CID and then triggered RDD within the peptide backbone. Our group later demonstrated that methyl branching, cyclopropane, and hydroxyl modifications could be localized upon CID of TPN-derivatized phosphatidylethanolamine ions. , In this study, we further expanded the analytical scope of CID-triggered RDD via TPN derivatization for the analysis of sphingoid bases. This method was integrated onto an RPLC-MS/MS workflow to realize the identification and relative quantitation of sphingoid bases hydrolyzed from bovine heart and Nematoda C. elegans .…”

“…Barrientos et al proposed that both electronic and steric effects played roles in OzID of the 4E CC in glycosphingolipids . Regarding the PB reaction, steric hindrance posed by the headgroup might be the main reason because the oxidized PE containing an OH group at the α-position of a CC away from the headgroup showed comparable PB reaction yield to an unoxidized PE . More recently, by combining MS with cryogenic gas-phase infrared spectroscopy (cryoIR), the Pagel group achieved detailed structural assignment of standard glycosphingolipids including the identity of the glycan, α- or β-glycosidic linkage in the glycan, and the lipid moiety including sphingosine and phytosphingosine …”

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis

“…While mass spectrometry based protocols provide exceptional sensitivity, specificity, and the ability to detect and quantify a wide range of lipid species involved in ferroptosis [ 281 ], they require specialized equipment, expertise, and sample preparation. The latest breakthroughs employing high-resolution mass spectrometry in conjunction with ion mobility and cutting-edge tandem mass spectrometry techniques have additionally enabled the direct identification of intact glycerophospholipids that bear oxidative labeling [ 282 , 283 ]. These advancements provide specific insights into the precise localization of these oxidative modifications [ 282 , 283 ].…”

“…The latest breakthroughs employing high-resolution mass spectrometry in conjunction with ion mobility and cutting-edge tandem mass spectrometry techniques have additionally enabled the direct identification of intact glycerophospholipids that bear oxidative labeling [ 282 , 283 ]. These advancements provide specific insights into the precise localization of these oxidative modifications [ 282 , 283 ]. Thiobarbituric acid reactive substances (TBARS) assay: The TBARS assay, which can be performed using commercial kits, is used to measure the levels of malondialdehyde (MDA), a product of lipid peroxidation [ 284 ].…”

First Proof of Concept of a Click Inverse Electron Demand Diels–Alder Reaction for Assigning the Regiochemistry of Carbon–Carbon Double Bonds in Untargeted Lipidomics

“…The proposed derivatization reactions introduce functional groups in correspondence with the CC bonds based on the reactivity of alkenes that facilitate diagnostic cleavages, i.e., epoxidation with meta -chloroperoxybenzoic acid . The group of Xia first reported the application of the Paternò–Büchi (PB) photocycloaddition reaction with acetone for assigning the regiochemistry of CC bonds in untargeted lipidomics. , Several effective PB reactions have been proposed for improved conversion efficiency, compatibility with MS imaging, , enhanced reactivity under visible light, or compatibility with radical-directed dissociation . Our research group has recently proposed an aza-Paternò–Büchi (aPB) reaction complementary to the PB reaction that enables regiochemistry assignment in the negative ion mode (ESI−) rather than in positive polarity (ESI+). , One of the current issues related to PB and aPB is due to the multitude of isomers that derive from the photocycloaddition, that result in broad and split peaks with poor peak shapes when reversed-phase (RP) LC separation before HRMS is employed. , The peak splitting is mainly due to the inherent asymmetry of PB and aPB reagents; thus, different approaches would be needed to overcome this issue.…”