Canonical fatty acid metabolism describes specific enzyme-substrate interactions that result in products with well-defined chain lengths, degree(s), and positions of unsaturation. Deep profiling lipids across a range prostate cancer cell lines reveals variety acids unusual site(s) unsaturation are not described by canonical pathways. The structure abundance these correlate changes desaturase expression strong indicators cellular phenotype. Gene silencing stable isotope tracing demonstrate...

Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity, and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells highly sensitive to ferroptosis, a form regulated death that is caused by oxidative stress through excess levels iron-dependent peroxidation polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity remain be...

Mass spectrometry imaging (MSI) of lipids within tissues has significant potential for both biomolecular discovery and histopathological applications. Conventional MSI technologies are, however, challenged by the prevalence phospholipid regioisomers that differ only in location(s) carbon–carbon double bonds and/or relative position fatty acyl attachment to glycerol backbone (i.e., sn position). The inability resolve isomeric experiments masks underlying complexity, resulting a critical loss...

Abstract Fatty acid isomers are responsible for an under-reported lipidome diversity across all kingdoms of life. Isomers unsaturated fatty acids often masked in contemporary analysis by incomplete separation and the absence sufficiently diagnostic methods structure elucidation. Here, we introduce a comprehensive workflow, to discover through coupling liquid chromatography mass spectrometry with gas-phase ozonolysis double bonds. The workflow encompasses semi-automated data enables de novo...

Abstract Osteoarthritis (OA) is a debilitating degenerative disease affecting multiple joint tissues, including cartilage, bone, synovium, and adipose tissues. OA presents diverse clinical phenotypes distinct molecular endotypes, inflammatory, metabolic, mechanical, genetic, synovial variants. Consequently, innovative technologies are needed to support the development of effective diagnostic precision therapeutic approaches. Traditional analysis bulk tissue extracts has limitations due...

Over 1500 different lipids have been reported in human plasma at the sum composition level. Yet number of unique present is surely higher, once isomeric contributions from double bond location(s) and fatty acyl regiochemistry are considered. In order to resolve this ambiguity, herein, we describe incorporation ozone-induced dissociation (OzID) into data-independent shotgun lipidomics workflows on a high-resolution hybrid quadrupole-Orbitrap platform. configuration, [M + Na]+ ions generated...

Acquired drug resistance is the major cause for disease recurrence in cancer patients, and this particularly true patients with metastatic melanoma that carry a BRAF

Separation and identification of fatty acid (FA) isomers in biological samples represents a challenging problem for lipid chemists. Notably, FA regio- stereo-isomers differing the location or (cis/trans) geometry carbon-carbon double bonds are often incompletely separated ambiguously assigned conventional chromatography-mass spectrometry analyses. To address this challenge, FAs have been derivatized with charge-switch derivatization reagents N-methyl-pyridinium-3-methanamine...

3-Pyridylcarbinol ester derivatization introduces a universal probe for cryogenic gas-phase infrared spectroscopy of fatty acids that enables assignment the position and configuration carbon–carbon double bonds even in presence isomers.

In recent years there has been a significant interest in the development of innovative lipidomics techniques capable resolving lipid isomers. To date, methods applied to sn-isomers have resolved only limited number species. We report workflow based on ozone-induced dissociation for untargeted characterisation hundreds sn-resolved glycerophospholipid isomers from biological extracts under 20 min, coupled with an automated data analysis pipeline. It provides order magnitude increase sn-isomer...

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake extracellular FAs their de novo synthesis. Given that oxidation synthesized for would result in net-energy loss, there is an implication from these two sources must have distinct metabolic fates; however, hitherto, all been considered part common pool. To probe potential partitioning FAs, cells were supplemented with stable isotope-labeled FAs. Structural analysis the resulting glycerophospholipids...

The specific positions of carbon–carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties lipid that ultimately inform its biological function(s). Contemporary liquid chromatography–mass spectrometry (MS) strategies based electrospray ionization coupled to tandem MS can easily detect acyl lipids but generally cannot reveal those site(s) unsaturation. Herein, we describe novel versatile workflow whereby acids are first converted...

Abstract In recent years there has been a significant interest in the development of innovative lipidomics techniques capable resolving lipid isomers. To date, methods applied to sn ‐isomers have resolved only limited number species. We report workflow based on ozone‐induced dissociation for untargeted characterisation hundreds sn‐ glycerophospholipid isomers from biological extracts under 20 min, coupled with an automated data analysis pipeline. It provides order magnitude increase isomer...

Abstract Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from precursor upon collision-induced dissociation (CID). It has been established that other classes of asymmetric glycerophospholipids, unimolecular fragmentation CID biased depending on relative position (known as sn-position) each chain...

Prostate cancer is the fourth most common worldwide with definitive diagnosis reliant on biopsy and human-graded histopathology. As other pathologies, grading based classical haematoxylin eosin (H&E) staining of formalin fixed paraffin-embedded material can be prone to variation between pathologists, prompting investigation biomolecular markers. Comprising around 50% cellular mass, known metabolic variations in cancer, lipids provide a promising target for molecular pathology. Here we apply...

Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry approaches, leading to a significant underestimation the structural diversity within lipidome. While ion mobility coupled mass has provided an additional dimension isomer resolution, some require resolving power beyond capabilities conventional platforms. Here, we present application high-resolution traveling-wave for separation that differ in (i) location single carbon-carbon double...

The onset and progression of cancer is associated with changes in the composition lipidome. Therefore, better understanding molecular mechanisms these disease states requires detailed structural characterization individual lipids within complex cellular milieu. Recently, unsaturation profile membrane have been observed cells tissues, but assigning position(s) carbon-carbon double bonds fatty acyl chains carried by phospholipids, including resolution lipid regioisomers, has proven...

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide valuable insights into the metabolome of complex biological systems such as organ tissues and cells. However, obtaining metabolite data at single-cell spatial resolutions presents a few technological challenges. Generally, resolution is defined by increment sample stage moves between ablation spots. Stage movements less than diameter focused beam (

Background: Enzymes are central components of many physiological processes, and changes in enzyme activity linked to numerous disease states, including osteoarthritis (OA). Assessing function can be challenging because difficulties separating affected tissue areas that result the homogenisation healthy diseased cells. Direct correlation between spatially-resolved distribution(s) cells/tissues thus lead advances our understanding OA pathophysiology. Herein, we present a method uses mass...

Gas-phase ion–molecule reactions provide structural insights across a range of analytical applications. A hindrance to the wider use is that they are relatively slow compared other ion activation modalities and can thereby impose bottleneck on time required analyze each sample. Here we describe method for accelerating rate involving ozone, implemented by supplementary RF-activation mass-selected ions within linear trap. Reaction accelerations between 15-fold (for ozonolysis alkenes in...

Abstract Acquired drug-resistance is a recurring problem in cancer treatment, and this particularly true for patients with metastatic melanoma that carry BRAF V600E mutation. In the current study, we explored use of membrane-active peptides as an alternative therapeutic modality to target drug-resistant cells. We produced slow-cycling cells using dabrafenib, small molecule drug targets tumor mutation, characterised their lipidome proteome investigate role membrane lipids acquired...

Abstract Background Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells highly sensitive to ferroptosis, a form regulated death that is caused by oxidative stress through excess levels iron-dependent peroxidation polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity...

ABSTRACT Fatty acid isomers are responsible for an under-reported lipidome diversity across all kingdoms of life. Isomers unsaturated fatty often masked in contemporary analysis by incomplete separation and the absence sufficiently diagnostic methods structure elucidation. Here, we introduce a comprehensive workflow to discover new acids through coupling liquid chromatography mass spectrometry with gas-phase ozonolysis double bonds. The encompasses semi-automated data enables de novo...