Gas-Phase Phenyl Radical + O2 Reacts via a Submerged Transition State.

In the gas-phase chemistry of the atmosphere and automotive fuel combustion, peroxyl radical intermediates are formed following O2 addition to carbon-centered radicals which then initiate a complex network of radical reactions that govern the oxidative processing of hydrocarbons. The rapid association of the phenyl radical-a fundamental radical related to benzene-with O2 has hitherto been modeled as a barrierless process, a common assumption for peroxyl radical formation. Here, we provide an alternate explanation for the kinetics of this reaction by deploying double-hybrid density functional theory (DFT), at the DSD-PBEP86-D3(BJ)/aug-cc-pVTZ level of theory, and locate a submerged adiabatic transition state connected to a prereaction complex along the reaction entrance pathway. Using this potential energy scheme, experimental rate coefficients k(T) for the addition of O2 to the phenyl radical are accurately reproduced within a microcanonical kinetic model. This work highlights that purportedly barrierless radical oxidation reactions may instead be modeled using stationary points, which in turn provides insight into pressure and temperature dependence.

Deep Characterisation of the sn-Isomer Lipidome Using High-Throughput Data-Independent Acquisition and Ozone-Induced Dissociation.

In recent years there has been a significant interest in the development of innovative lipidomics techniques capable of resolving lipid isomers. To date, methods applied to resolving sn-isomers have resolved only a limited number of species. We report a workflow based on ozone-induced dissociation for untargeted characterisation of hundreds of sn-resolved glycerophospholipid isomers from biological extracts in under 20 min, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn-isomer pairs identified as compared to previous reports and reveals that sn-isomer populations are tightly regulated and significantly different between cell lines. The sensitivity of this method and potential for de novo molecular discovery is further demonstrated by the identification of unexpected lipids containing ultra-long monounsaturated acyl chains at the sn-1 position.

Peptide Self-Assembly Controlled Photoligation of Polymers.

Highly efficient chemical ligations that operate in water under mild conditions are the foundation of bioorthogonal chemistry. However, the toolbox of suitable reactions is limited. Conventional approaches to expand this toolbox aim at altering the inherent reactivity of functional groups to design new reactions that meet the required benchmarks. Inspired by controlled reaction environments that enzymes provide, we report a fundamentally different approach that makes inefficient reactions highly efficient within defined local environments. Contrasting enzymatically catalyzed reactions, the reactivity controlling self-assembled environment is brought about by the ligation targets themselves─avoiding the use of a catalyst. Targeting [2 + 2] photocycloadditions, which are inefficient at low concentrations and readily quenched by oxygen, short ß-sheet encoded peptide sequences are inserted between a hydrophobic photoreactive styrylpyrene unit and a hydrophilic polymer. In water, electrostatic repulsion of deprotonated amino acid residues governs the formation of small self-assembled structures, which enable a highly efficient photoligation of the polymer, reaching ∼90% ligation within 2 min (0.034 mM). Upon protonation at low pH, the self-assembly changes into 1D fibers, altering photophysical properties and shutting down the photocycloaddition reaction. Using the reversible morphology change, it is possible to switch the photoligation "ON" or "OFF" under constant irradiation simply by varying the pH. Importantly, in dimethylformamide, the photoligation reaction did not occur even at 10-fold higher concentrations (0.34 mM). The self-assembly into a specific architecture, encoded into the polymer ligation target, enables a highly efficient ligation that overcomes the concentration limitations and high oxygen sensitivity of [2 + 2] photocycloadditions.

Revolutions in Lipid Isomer Resolution: Application of Ultrahigh-Resolution Ion Mobility to Reveal Lipid Diversity.

Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry approaches, leading to a significant underestimation of the structural diversity within the lipidome. While ion mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require a resolving power beyond the capabilities of conventional platforms. Here, we present the application of high-resolution traveling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single carbon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans), or, for glycerolipids, (iii) the relative substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility separation allowed identification of the carbon-carbon double-bond position and sn-position, enabling confident interpretation of variations in mobility peak abundance. To demonstrate the applicability of this method, double-bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of up to six isomers. These findings suggest that ultrahigh-resolution ion mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.

Programming Photodegradability into Vinylic Polymers via Radical Ring-Opening Polymerization.

Incorporation of photolabile moieties into the polymer backbone holds promise to remotely-control polymer degradation. However, suitable synthetic avenues are limited, especially for radical polymerizations. Here we report a strategy to program photodegradability into vinylic polymers by exploiting the wavelength selectivity of photocycloadditions for radical ring-opening polymerization (rROP). Irradiation of coumarin terminated allylic sulfides with UVA light initiated intramolecular [2+2] photocycloaddition producing cyclic macromonomers. Subsequent RAFT-mediated rROP with methyl acrylate yielded copolymers that inherited the photoreactivity of the cyclic parent monomer. Irradiation with UVB initiated efficient photocycloreversion of the coumarin dimers, causing polymer degradation within minutes under UVB light or days under sunlight exposure. Our synthetic strategy may pave the way to insert photolabile linkages into vinylic polymers, tuning degradation for specific wavelengths.

Diastereomer Resolution of M4 L6 Coordination Cages by Ultra-High-Resolution Ion-Mobility Mass Spectrometry.

Coordination cages can be used for enantio- and regioselective catalysis and for the selective sensing and separation of isomeric guest molecules. Here, stereoisomers of a family of coordination cages are resolved using ultra-high-resolution cyclic ion-mobility mass spectrometry (cIM-MS). The observed ratio of diastereomers is dependent on both the metal ion and counter ion. Moreover, the point groups can be assigned through complementary NMR experiments. This method enables the identification and interrogation of the individual isomers in complex mixtures of cages which cannot be performed in solution. Furthermore, these techniques allow the stability of individual isomers within the mixture to be probed, with the T-symmetric isomers in this case shown to be more robust than the C3 and S4 analogues.

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Accelerating Ozonolysis Reactions Using Supplemental RF-Activation of Ions in a Linear Ion Trap Mass Spectrometer.

Gas-phase ion-molecule reactions provide structural insights across a range of analytical applications. A hindrance to the wider use of ion-molecule reactions is that they are relatively slow compared to other ion activation modalities and can thereby impose a bottleneck on the time required to analyze each sample. Here we describe a method for accelerating the rate of ion-molecule reactions involving ozone, implemented by supplementary RF-activation of mass-selected ions within a linear ion trap. Reaction rate accelerations between 15-fold (for ozonolysis of alkenes in ionised lipids) and 90-fold (for ozonation of halide anions) are observed compared to thermal conditions. These enhanced reaction rates with ozone increase sample throughput, aligning the reaction time with the overall duty cycle of the mass spectrometer. We demonstrate that the acceleration is due to the supplementary RF-activation surmounting the activation barrier energy of the entrance channel of the ion-molecule reaction. This rate acceleration is subsequently shown to aid identification of new, low abundance lipid isomers and enables an equivalent increase in the number of lipid species that can be analyzed.

Identification of Carbon-Carbon Double Bond Stereochemistry in Unsaturated Fatty Acids by Charge-Remote Fragmentation of Fixed-Charge Derivatives.

Separation and identification of fatty acid (FA) isomers in biological samples represents a challenging problem for lipid chemists. Notably, FA regio- and stereo-isomers differing in the location or (cis/trans) geometry of carbon-carbon double bonds are often incompletely separated and ambiguously assigned in conventional chromatography-mass spectrometry analyses. To address this challenge, FAs have been derivatized with the charge-switch derivatization reagents N-methyl-pyridinium-3-methanamine and N-(4-aminomethylphenyl)pyridinium and subjected to reversed-phase liquid chromatography-tandem mass spectrometry. Charge-remote fragmentation of the fixed-charge derivatives leads to characteristic product ions arising from dissociation at allylic positions that enable assignment of position(s) of unsaturation, while a newly discovered dihydrogen neutral loss was found to be dominant for double bonds with cis-stereochemistry. The structure of the [M - 2]+ product ions was probed by gas-phase ozonolysis revealing the presence of two new carbon-carbon bonds on either side of the initial position of unsaturation consistent with an electrocyclic mechanism of 1,4-dihydrogen elimination. Charge-remote fragmentation pathways diagnostic of double bond position and stereochemistry were found to be generalized for FAs of different carbon-chain lengths, double bond positions, and degrees of unsaturation and were effective in the unequivocal assignment of the FA structure in complex mixtures of FA isomers, including bovine milk powder.

Exploring the Gas-Phase Formation and Chemical Reactivity of Highly Reduced M8 L6 Coordination Cages.

Coordination cages with well-defined cavities show great promise in the field of catalysis on account of their unique combination of molecular confinement effects and transition-metal redox chemistry. Here, three coordination cages are reduced from their native 16+ oxidation state to the 2+ state in the gas phase without observable structural degradation. Using this method, the reaction rate constants for each reduction step were determined, with no noticeable differences arising following either the incorporation of a C60 -fullerene guest or alteration of the cage chemical structure. The reactivity of highly reduced cage species toward molecular oxygen is "switched-on" after a threshold number of reduction steps, which is influenced by guest molecules and the structure of cage components. These new experimental approaches provide a unique window to explore the chemistry of highly-reduced cage species that can be modulated by altering their structures and encapsulated guest species.

Mass Spectrometry Imaging of Lipids with Isomer Resolution Using High-Pressure Ozone-Induced Dissociation.

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 of phospholipid regioisomers that differ only in the location(s) of carbon-carbon double bonds and/or the relative position of fatty acyl attachment to the glycerol backbone (i.e., sn position). The inability to resolve isomeric lipids within imaging experiments masks underlying complexity, resulting in a critical loss of metabolic information. Herein, ozone-induced dissociation (OzID) is implemented on a mobility-enabled quadrupole time-of-flight (Q-TOF) mass spectrometer capable of matrix-assisted laser desorption/ionization (MALDI). Exploiting the ion mobility region in the Q-TOF, high number densities of ozone were accessed, leading to ∼1000-fold enhancement in the abundance of OzID product ions compared to earlier MALDI-OzID implementations. Translation of this uplift into imaging resulted in a 50-fold improvement in acquisition rate, facilitating large-area mapping with resolution of phospholipid isomers. Mapping isomer distributions across rat brain sections revealed distinct distributions of lipid isomer populations with region-specific associations of isomers differing in double bond and sn positions. Moreover, product ions arising from sequential ozone- and collision-induced dissociation enabled double bond assignments in unsaturated fatty acyl chains esterified at the noncanonical sn-1 position.

Next-generation derivatization reagents optimized for enhanced product ion formation in photodissociation-mass spectrometry of fatty acids.

Ultraviolet-photodissociation (UVPD) mass spectrometry is an emerging analytical tool for structural elucidation of biomolecules including lipids. Gas phase UVPD of ionised fatty acids (FAs) can promote fragmentation that is diagnostic for molecular structure including the regiochemistry of carbon-carbon double bonds and methyl branching position(s). Typically, however, lipids exhibit poor conversion to photoproducts under UVPD and thus require longer integration times to achieve the signal-to-noise required for structural assignments. Consequently, the integration of UVPD into liquid-chromatography mass spectrometry (LC-MS) workflows for FAs has been limited. To enhance photofragmentation efficiency, an alternative strategy has been devised using wet-chemical derivatization of FAs to explicitly incorporate photolabile groups. FA derivatives that include an aryl-iodide motif have photodissociation conversions of up to 28% when activated by a single 266 nm photon. The radical-directed dissociation product ions resulting from UVPD of these derivatives provide key details of molecular structure and discriminate between lipid isomers. Herein, we describe the structure-activity guided development of new FA derivatives capable of photoproduct yields of up to 97%. UVPD-action spectroscopy demonstrates that photodissociation for FAs derivatized with N-(2-aminoethyl)-4-iodobenzamide (NIBA) is maximised near 266 nm and highlights the key role of the 4-iodobenzamide motif in the efficient formation of [M - I]˙+ radical cations (and diagnostic secondary product ions). The high photodissociation yield of NIBA-derivatized lipids is maintained across 37 commonly observed FAs with the resulting UVPD mass spectra shown to be effective in the discrimination of isomeric FAs that differ in the position(s) of carbon-carbon double bonds. Integration of this strategy with reversed-phase LC-MS workflows is confirmed with high-quality UVPD mass spectra acquired across each chromatographic peak.

Structural elucidation of hydroxy fatty acids by photodissociation mass spectrometry with photolabile derivatives.

RATIONALE: Eicosanoids are short-lived bio-responsive lipids produced locally from oxidation of polyunsaturated fatty acids (FAs) via a cascade of enzymatic or free radical reactions. Alterations in the composition and concentration of eicosanoids are indicative of inflammation responses and there is strong interest in developing analytical methods for the sensitive and selective detection of these lipids in biological mixtures. Most eicosanoids are hydroxy FAs (HFAs), which present a particular analytical challenge due to the presence of regioisomers arising from differing locations of hydroxylation and unsaturation within their structures. METHODS: In this study, the recently developed derivatization reagent 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+ ) was applied to a representative set of HFAs including bioactive eicosanoids. Photodissociation (PD) mass spectra obtained at 266 nm of 4-I-AMPP+ -modified HFAs exhibit abundant product ions arising from photolysis of the aryl-iodide bond within the derivative with subsequent migration of the radical to the hydroxyl group promoting fragmentation of the FA chain and facilitating structural assignment. RESULTS: Representative polyunsaturated HFAs (from the hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid families) were derivatized with 4-I-AMPP+ and subjected to a reversed-phase liquid chromatography workflow that afforded chromatographic resolution of isomers in conjunction with structurally diagnostic PD mass spectra. CONCLUSIONS: PD of these complex HFAs was found to be sensitive to the locations of hydroxyl groups and carbon-carbon double bonds, which are structural properties strongly associated with the biosynthetic origins of these lipid mediators.

Gas phase reactions of iodide and bromide anions with ozone: evidence for stepwise and reversible reactions.

Despite the impacts - both positive and negative - of atmospheric ozone for life on Earth, there remain significant gaps in our knowledge of the products, mechanisms and rates of some of its most fundamental gas phase reactions. This incomplete understanding is largely due to the experimental challenges involved in the study of gas-phase reactions of ozone and, in particular, the identification of short-lived reaction intermediates. Here we report direct observation of the stepwise reaction of the halide anions iodide (I-) and bromide (Br-) with ozone to produce XO3- (where X = I and Br, respectively). These results substantially revise the rate constant for the I- + O3 reaction to 1.1 (± 0.5) × 10-12 cm3 molecule-1 s-1 (0.13% efficiency) and the Br- + O3 reaction to 6.2 (± 0.4) × 10-15 cm3 molecule-1 s-1 (0.001% efficiency). Exploiting five-orders of temporal dynamic range on a linear ion trap mass spectrometer enabled explicit measurement of the rate constants for the highly efficient intermediate, XO- + O3 and XO2- + O3, reactions thus confirming a stepwise addition of three oxygen atoms (i.e., X- + 3O3 → XO3- + 3O2) with the first addition representing the rate determining step. Evidence is also presented for (i) slow reverse reactions of XO- and XO2-, but not XO3-, with molecular oxygen and (ii) the photodissociation of IO-, IO2- and IO3- to release I-. Collectively, these results suggest relatively short lifetimes for Br- and I- in the tropospere with direct gas-phase oxidation by ozone playing a role in both the formation of atmospheric halogen oxides and, conversely, in the ozone depletion associated with springtime polar bromine explosion events.

Resolving Sphingolipid Isomers Using Cryogenic Infrared Spectroscopy.

1-Deoxysphingolipids are a recently described class of sphingolipids that have been shown to be associated with several disease states including diabetic and hereditary neuropathy. The identification and characterization of 1-deoxysphingolipids and their metabolites is therefore highly important. However, exact structure determination requires a combination of sophisticated analytical techniques due to the presence of various isomers, such as ketone/alkenol isomers, carbon-carbon double-bond (C=C) isomers and hydroxylation regioisomers. Here we demonstrate that cryogenic gas-phase infrared (IR) spectroscopy of ionized 1-deoxysphingolipids enables the identification and differentiation of isomers by their unique spectroscopic fingerprints. In particular, C=C bond positions and stereochemical configurations can be distinguished by specific interactions between the charged amine and the double bond. The results demonstrate the power of gas-phase IR spectroscopy to overcome the challenge of isomer resolution in conventional mass spectrometry and pave the way for deeper analysis of the lipidome.

Analytical separations for lipids in complex, nonpolar lipidomes using differential mobility spectrometry.

Secretions from meibomian glands located within the eyelid (commonly known as meibum) are rich in nonpolar lipid classes incorporating very-long (22-30 carbons) and ultra-long (>30 carbons) acyl chains. The complex nature of the meibum lipidome and its preponderance of neutral, nonpolar lipid classes presents an analytical challenge, with typically poor chromatographic resolution, even between different lipid classes. To address this challenge, we have deployed differential mobility spectrometry (DMS)-MS to interrogate the human meibum lipidome and demonstrate near-baseline resolution of the two major nonpolar classes contained therein, namely wax esters and cholesteryl esters. Within these two lipid classes, we describe ion mobility behavior that is associated with the length of their acyl chains and location of unsaturation. This capability was exploited to profile the molecular speciation within each class and thus extend meibum lipidome coverage. Intriguingly, structure-mobility relationships in these nonpolar lipids show similar trends and inflections to those previously reported for other physicochemical properties of lipids (e.g., melting point and phase-transition temperatures). Taken together, these data demonstrate that differential ion mobility provides a powerful orthoganol separation technology for the analysis of neutral lipids in complex matrices, such as meibum, and may further provide a means to predict physicochemical properties of lipids that could assist in inferring their biological function(s).

Introduction of a Fixed-Charge, Photolabile Derivative for Enhanced Structural Elucidation of Fatty Acids.

Fatty acids are a structurally diverse category of lipids with a myriad of biochemical functions, which includes their role as building blocks of more complex lipids (e.g., glycerophospholipids and triacylglycerols). Increasingly, the analysis of fatty acids is undertaken using liquid chromatography-mass spectrometry (LC-MS), due to its versatility in the detection of lipids across a wide range of concentrations and diversity of molecular structures and masses. Previous work has shown that fixed-charge pyridinium derivatives are effective in enhancing the detection of fatty acids in LC-MS workflows. Herein, we describe the development of two novel pyridinium fixed-charged derivatization reagents that incorporate a photolabile aryl iodide that is selectively activated by laser irradiation inside the mass spectrometer. Photodissociation mass spectra of fatty acids conjugated to 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) and 1-(4-(aminomethyl)-3-iodophenyl)pyridin-1-ium (3-I-AMPP+) derivatives reveal structurally diagnostic product ions. These spectra feature radical-directed dissociation of the carbon-carbon bonds within the fatty acyl chain, enabling structural assignments of fatty acids and discrimination of isomers that differ in site(s) of unsaturation, methyl branching or cyclopropanation. These derivatives are shown to be suitable for hyphenated LC-MS methods, and their predictable photodissociation behavior allows de novo identification of unusual fatty acids within a biological context.

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids.

The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.

Online Ozonolysis Combined with Ion Mobility-Mass Spectrometry Provides a New Platform for Lipid Isomer Analyses.

One of the most significant challenges in contemporary lipidomics lies in the separation and identification of lipid isomers that differ only in site(s) of unsaturation or geometric configuration of the carbon-carbon double bonds. While analytical separation techniques including ion mobility spectrometry (IMS) and liquid chromatography (LC) can separate isomeric lipids under appropriate conditions, conventional tandem mass spectrometry cannot provide unequivocal identification. To address this challenge, we have implemented ozone-induced dissociation (OzID) in-line with LC, IMS, and high resolution mass spectrometry. Modification of an IMS-capable quadrupole time-of-flight mass spectrometer was undertaken to allow the introduction of ozone into the high-pressure trapping ion funnel region preceding the IMS cell. This enabled the novel LC-OzID-IMS-MS configuration where ozonolysis of ionized lipids occurred rapidly (10 ms) without prior mass-selection. LC-elution time alignment combined with accurate mass and arrival time extraction of ozonolysis products facilitated correlation of precursor and product ions without mass-selection (and associated reductions in duty cycle). Unsaturated lipids across 11 classes were examined using this workflow in both positive and negative ion modalities, and in all cases, the positions of carbon-carbon double bonds were unequivocally assigned based on predictable OzID transitions. Under these conditions, geometric isomers exhibited different IMS arrival time distributions and distinct OzID product ion ratios providing a means for discrimination of cis/trans double bonds in complex lipids. The combination of OzID with multidimensional separations shows significant promise for facile profiling of unsaturation patterns within complex lipidomes including human plasma.

Differential-Mobility Spectrometry of 1-Deoxysphingosine Isomers: New Insights into the Gas Phase Structures of Ionized Lipids.

Separation and structural identification of lipids remain a major challenge for contemporary lipidomics. Regioisomeric lipids differing only in position(s) of unsaturation are often not differentiated by conventional liquid chromatography-mass spectrometry approaches leading to the incomplete, or sometimes incorrect, assignation of molecular structure. Here we describe an investigation of the gas phase separations by differential-mobility spectrometry (DMS) of a series of synthetic analogues of the recently described 1-deoxysphingosine. The dependence of the DMS behavior on the position of the carbon-carbon double bond within the ionized lipid is systematically explored and compared to trends from complementary investigations, including collision cross-sections measured by drift tube ion mobility, reaction efficiency with ozone, and molecular dynamics simulations. Consistent trends across these modes of interrogation point to the importance of direct, through-space interactions between the charge site and the carbon-carbon double bond. Differences in the geometry and energetics of this intramolecular interaction underpin DMS separations and influence reactivity trends between regioisomers. Importantly, the disruption and reformation of these intramolecular solvation interactions during DMS are proposed to be the causative factor in the observed separations of ionized lipids which are shown to have otherwise identical collision cross-sections. These findings provide key insights into the strengths and limitations of current ion-mobility technologies for lipid isomer separations and can thus guide a more systematic approach to improved analytical separations in lipidomics.