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.

9-Azahomocubane.

Homocubane, a highly strained cage hydrocarbon, contains two very different positions for the introduction of a nitrogen atom into the skeleton, e. g., a position 1 exchange results in a tertiary amine whereas position 9 yields a secondary amine. Herein reported is the synthesis of 9-azahomocubane along with associated structural characterization, physical property analysis and chemical reactivity. Not only is 9-azahomocubane readily synthesized, and found to be stable as predicted, the basicity of the secondary amine was observed to be significantly lower than the structurally related azabicyclo[2.2.1]heptane, although similar to 1-azahomocubane.

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.

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.

1-Azahomocubane.

Highly strained cage hydrocarbons have long stood as fundamental molecules to explore the limits of chemical stability and reactivity, probe physical properties, and more recently as bioactive molecules and in materials discovery. Interestingly, the nitrogenous congeners have attracted much less attention. Previously absent from the literature, azahomocubanes, offer an opportunity to investigate the effects of a nitrogen atom when incorporated into a highly constrained polycyclic environment. Herein disclosed is the synthesis of 1-azahomocubane, accompanied by comprehensive structural characterization, physical property analysis and chemical reactivity. These data support the conclusion that nitrogen is remarkably well tolerated in a highly strained environment.

Electrochemical degradation and mineralisation of organic dyes in aqueous nitrate solutions.

Electrochemical treatment of organic matter for environmental remediation necessitates the development of cheap and robust electrodes that are chemically and structurally stable. To address this challenging requirement, we demonstrate a new electrochemical approach using a simple copper electrode under cathodic conditions to electrochemically generate reactive nitrosonium ions for the degradation of different classes of synthetic organic dyes. This could be achieved in an aqueous HNO3/KNO3 electrolyte at a relatively low cathodic potential of -0.5 V RHE at room temperature. UV-visible absorption spectroscopy, Raman spectroscopy, liquid chromatography - mass spectrometry and total organic carbon measurements revealed the rapid decolorisation and mineralisation of several dye types such as triarylmethane dyes (crystal violet, cresol red), an azo dye (methyl orange) as well as a sulfur containing thiazine dye (toluidine blue). The total organic carbon content of a 50 mg L-1 methyl orange solution was found to decrease by 83% after 1 h of electrolysis. Promisingly, locally sourced river and creek water samples spiked with 50 mg L-1 methyl orange were also successfully treated for up to 6 cycles at a simple Cu electrode, demonstrating potential for the remediation of polluted waterways.

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.

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.

Reference intervals for hematological and biochemical analytes in a single herd of clinically healthy gelding donkeys in Saint Kitts.

There is a large population of donkeys in Saint Kitts; however, hematological and biochemical reference intervals (RIs) are lacking. This study addressed this deficiency by following the American Society for Veterinary Clinical Pathology RI guidelines. Sixty-six healthy, gelding standard donkeys with a median and interquartile range age of 5 years (3.5 - 8 years) and a mean ± standard deviation body weighed of 156 ± 16.7 kg were used to produce a five-part differential complete blood count using an impedance-based analyzer. Clinical chemistry analytes were quantified using a photometric-based analyzer utilizing two reagent rotors that determined 14 and 11 analytes, respectively. An electrochemical-based analyzer quantified chloride, sodium and potassium. Reference intervals were computed using Reference Value Advisor. Results of analytes determined using different rotors/analyzers were assessed using Passing-Bablok regression and Bland-Altman plot analyses. Reference intervals for 43 hematological and biochemical analytes were generated. Reference intervals for hematocrit, red blood cells, white blood cells, total protein, glucose, blood urea nitrogen, and creatinine were 23.67% - 38.08%, 4.08 - 6.42 1012/L, 4.7 - 12.34 109/L, 5.84 - 6.93 g/dL, 64.7 - 130.9 mg/dL, 11.1 - 13.4 mg/dL, and 0.67 - 1.36 mg/dL, respectively. There was good agreement between detection system for albumin, aspartate aminotransferase, gamma glutamyl transferase, total protein, globulin, and potassium, but not for blood urea nitrogen, calcium, creatinine kinase, and sodium. This study is the first to establish hematological and biochemical RIs in donkeys in Saint Kitts. These values will be useful for clinical decision-making.

Laser Photodissociation Action Spectroscopy for the Wavelength-Dependent Evaluation of Photoligation Reactions.

The nitrile imine-mediated tetrazole-ene cycloaddition is a widely used class of photoligation. Optimizing the reaction outcome requires detailed knowledge of the tetrazole photoactivation profile, which can only partially be ascertained from absorption spectroscopy, or otherwise involves laborious reaction monitoring in solution. Photodissociation action spectroscopy (PDAS) combines the advantages of optical spectroscopy and mass spectrometry in that only absorption events resulting in a mass change are recorded, thus revealing the desired wavelength dependence of product formation. Moreover, the sensitivity and selectivity afforded by the mass spectrometer enable reliable assessment of the photodissociation profile even on small amounts of crude material, thus accelerating the design and synthesis of next-generation substrates. Using this workflow, we demonstrate that the photodissociation onset for nitrile imine formation is red-shifted by ca. 50 nm with a novel N-ethylcarbazole derivative relative to a phenyl-substituted archetype. Benchmarked against solution-phase tunable laser experiments and supported by quantum chemical calculations, these discoveries demonstrate that PDAS is a powerful tool for rapidly screening the efficacy of new substrates in the quest toward efficient visible light-triggered ligation for biological applications.

Apocryphal FADS2 activity promotes fatty acid diversification in cancer.

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 of lipids across a range of prostate cancer cell lines reveals a variety of fatty acids with unusual site(s) of unsaturation that are not described by canonical pathways. The structure and abundance of these unusual lipids correlate with changes in desaturase expression and are strong indicators of cellular phenotype. Gene silencing and stable isotope tracing demonstrate that direct Δ6 and Δ8 desaturation of 14:0 (myristic), 16:0 (palmitic), and 18:0 (stearic) acids by FADS2 generate new families of unsaturated fatty acids (including n-8, n-10, and n-12) that have rarely-if ever-been reported in human-derived cells. Isomer-resolved lipidomics reveals the selective incorporation of these unusual fatty acids into complex structural lipids and identifies their presence in cancer tissues, indicating functional roles in membrane structure and signaling.

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.

Stepwise reduction of interlocked viologen-based complexes in the gas phase.

We present the first application of electrochemical reduction in an ion trap mass spectrometer as a dual-function tool to synthesise and probe the reactivity of interlocked viologen-based complexes. Compared with non-complexed archetypes, electron-donating macrocyclic porphyrin ethers retard electron transfer reaction rates and stabilise intact structures in low oxidation states.

Charge-switch derivatization of fatty acid esters of hydroxy fatty acids via gas-phase ion/ion reactions.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS2 product ion spectra of [FAHFA - H]¯ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen)32+, to selectively and efficiently derivatize [FAHFA - H]¯ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA - H + MgPhen2]+ cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards.

Phosphoproteomic Analysis across the Yeast Life Cycle Reveals Control of Fatty Acyl Chain Length by Phosphorylation of the Fatty Acid Synthase Complex.

The ability to remodel lipid metabolism under changing conditions is pivotal for cellular functionality and homeostasis. Here, we characterize the regulatory landscape of phosphorylation-based signaling events across the life cycle of Saccharomyces cerevisiae and determine its impact on the regulation of lipid metabolism. Our data show that 50 lipid metabolic proteins are differentially phosphorylated as cells transit between different physiological states. To identify functional phosphosites, we devised a strategy where multiple phosphosites are simultaneously mutated into phosphomimetic or phosphodeficient alleles and mutants are phenotyped by in-depth lipidomics flux analysis. This uncovers functional phosphosites in the phosphatidate cytidylyltransferase Cds1, the phosphatidylserine synthase Cho1, and Fas2, the α-subunit of the fatty acid synthase (FAS) complex. Furthermore, we show that the fatty acyl chain length produced by FAS is governed by phosphorylation. Overall, our work demonstrates a vital role for phosphoregulation of lipid metabolism and provides a resource to investigate its molecular underpinnings.

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.

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.

Mapping Enzyme Activity on Tissue by Functional Mass Spectrometry Imaging.

Enzymes are central components of most physiological processes, and are consequently implicated in various pathologies. High-resolution maps of enzyme activity within tissues therefore represent powerful tools for elucidating enzymatic functions in health and disease. Here, we present a novel mass spectrometry imaging (MSI) method for assaying the spatial distribution of enzymatic activity directly from tissue. MSI analysis of tissue sections exposed to phospholipid substrates produced high-resolution maps of phospholipase activity and specificity, which could subsequently be compared to histological images of the same section. Functional MSI thus represents a new and generalisable method for imaging biological activity in situ.

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.

Combining Charge-Switch Derivatization with Ozone-Induced Dissociation for Fatty Acid Analysis.

The specific positions of carbon-carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography-mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N-(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon-carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa. This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways.