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

Cerrato, Andrea; Cavaliere, Chiara; Laganà, Aldo; Montone, Carmela Maria; Piovesana, Susy; Sciarra, Alessandro; Taglioni, Enrico; Capriotti, Anna Laura

doi:10.1021/acs.analchem.4c02146

Cited by 3 publications

(1 citation statement)

References 61 publications

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“…To surmount these limitations, a number of alternative approaches have emerged targeting double bond positional assignment in complex lipids that can be broadly classified as (1) chemical derivatization of carbon–carbon double bonds prior to mass spectrometry in order to activate sites of unsaturation toward fragmentation upon CID, − and (2) alternative methods of ion activation to promote either (2a) cleavage of most carbon–carbon bonds where interruptions at sites of unsaturation can be identified, − or (2b) selective carbon–carbon bond cleavage at, or adjacent to, alkene positions. ,− Among several solution-phase derivatization strategies, including epoxidation and more recently aziridination and Diels–Alder reactions, − the most widely adopted approach is the Paternò-Büchi reaction that involves a [2 + 2] cycloaddition between the double bond and an appropriate ketone or aldehyde (e.g., acetone, benzophenone) under exposure to UV-light. ,− Once the cyclic oxetane products are formed in solution they are ionized, mass-selected and subjected to CID yielding two diagnostic product ions with aldehyde and trisubstituted alkene motifs. The characteristic spacing between these two marker ions (Δ m = 26.0520 Da when using acetone) in the resulting spectrum can be exploited for de novo assignment of the double bond position (Scheme A) .…”

Section: Introductionmentioning

confidence: 99%

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer

Brydon,

Poad,

Fang

et al. 2024

J. Am. Soc. Mass Spectrom.

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The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon−carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon−carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.

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Section: Introductionmentioning

confidence: 99%

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer

Brydon,

Poad,

Fang

et al. 2024

J. Am. Soc. Mass Spectrom.

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Determination of double bond positions in unsaturated fatty acids by pre-column derivatization with dimethyl and dipyridyl disulfide followed by LC-SWATH-MS analysis

Olfert,

Knappe,

Sievers-Engler

et al. 2024

Anal Bioanal Chem

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Comprehensive in-depth structural characterization of free mono-unsaturated and polyunsaturated fatty acids often requires the determination of carbon-carbon double bond positions due to their impact on physiological properties and relevance in biological samples or during impurity profiling of pharmaceuticals. In this research, we report on the evaluation of disulfides as suitable derivatization reagents for the determination of carbon-carbon double bond positions of unsaturated free fatty acids by UHPLC-ESI-QTOF-MS/MS analysis and SWATH (sequential windowed acquisition of all theoretical mass spectra) acquisition. Iodine-catalyzed derivatization of C = C double bonds with dimethyl disulfide (DMDS) enabled detection of characteristic carboxy-terminal MS2 fragments for various fatty acids in ESI negative mode. The determination of double bond positions of fatty acids with up to three double bonds, the transfer of the method to plasma samples, and its limitations have been shown. To achieve charge-switching for positive ion mode MS-detection, derivatization with 2,2′-dipyridyldisulfide (DPDS) was investigated. It enabled detection of both corresponding characteristic omega-end- and carboxy-end-fragments for fatty acids with up to two double bonds in positive ion mode. It provides a straightforward strategy for designing MRM transitions for targeted LC–MS/MS assays. Both derivatization techniques represent a simple and inexpensive way for the determination of double bond positions in fatty acids with low number of double bonds. No adaptation of MS hardware is required and the specific isotopic pattern of resulting sulfur-containing products provides additional structural confirmation. This reaction scheme opens up the avenue of structural tuning of disulfide reagents beyond DMDS and DPDS using reagents like cystine and analogs to achieve enhanced performance and sensitivity. Graphical Abstract

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Tandem Mass Spectrometry in Untargeted Lipidomics: A Case Study of Peripheral Blood Mononuclear Cells

Ventura,

Bianco,

Calvano

et al. 2024

IJMS

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Peripheral blood mononuclear cells (PBMCs), including lymphocytes, are important components of the human immune system. These cells contain a diverse array of lipids, primarily glycerophospholipids (GPs) and sphingolipids (SPs), which play essential roles in cellular structure, signaling, and programmed cell death. This study presents a detailed analysis of GP and SP profiles in human PBMC samples using tandem mass spectrometry (MS/MS). Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled with linear ion-trap MS/MS were employed to investigate the diagnostic fragmentation patterns that aided in determining regiochemistry in complex lipid extracts. Specifically, the study explored the fragmentation patterns of various lipid species, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), their plasmalogen and lyso forms, phosphatidylserines (PSs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs), sphingomyelins (SMs), and dihexosylceramides (Hex2Cer). Our comprehensive analysis led to the characterization of over 200 distinct lipid species, significantly expanding our understanding of PBMC lipidome complexity. A freely available spreadsheet tool for simulating MS/MS spectra of GPs is provided, enhancing the accessibility and reproducibility of this research. This study advances our knowledge of PBMC lipidomes and establishes a robust analytical framework for future investigations in lipidomics.

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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

Cited by 3 publications

References 61 publications

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer

Determination of double bond positions in unsaturated fatty acids by pre-column derivatization with dimethyl and dipyridyl disulfide followed by LC-SWATH-MS analysis

Tandem Mass Spectrometry in Untargeted Lipidomics: A Case Study of Peripheral Blood Mononuclear Cells

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