Individual Isomers Research Articles

Evaluation of the ionization efficiency in phosphatidylcholine positional isomers with docosahexaenoic acid bound to the sn-1 or sn-2 position

Phosphatidylcholine (PC), a key phospholipid, contains 2 fatty acids that can be bound at the sn-1 and sn-2 positions, resulting in positional isomers when different fatty acids are attached. Currently, there is no established method for identifying phospholipid molecular species and quantifying individual isomers using authentic standards of each PC isomer. In this study, we prepare authentic analytical standards for PC positional isomers through chemical synthesis and preparative purification. These isomers contain docosahexaenoic acid (DHA, 22:6) and palmitic acid (16:0) attached at the sn-1 and sn-2 positions and are denoted as PC(22:6/16:0) and PC(16:0/22:6), respectively. Standard solutions of PC(22:6/16:0) and PC(16:0/22:6) were analyzed using liquid chromatography-tandem mass spectrometry, and calibration curves of the PC positional isomers were generated to compare their ionization efficiencies. The ionization efficiency of PC(22:6/16:0) was 2.32 times higher than that of PC(16:0/22:6), indicating that the ionization efficiency depends on the binding position of the fatty acid. Elucidating and correcting the differences in the ionization efficiencies of the PC positional isomers will enable the accurate quantitative analysis of lipidomes in the future.

Utilizing ESI-MS/MS and ion mobility spectrometry for structural characterization and isomer differentiation of 1, 2-unsaturated (1-4) linked disaccharide derivatives and their 2-C-functionalized analogues.

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

Cyclic Ion Mobility of Isomeric New Psychoactive Substances Employing Characteristic Arrival Time Distribution Profiles and Adduct Separation.

Analysis of new psychoactive substances (NPS), which is essential for toxicological and forensic reasons, can be made complicated by the presence of isomers. Ion mobility has been used as a standalone technique or coupled to mass spectrometry to detect and identify NPS. However, isomer separation has so far chiefly relied on chromatography. Here we report on the determination of isomeric ratios using cyclic ion mobility-mass spectrometry without any chromatographic separation. Isomers were distinguished by mobility separation of lithium adducts. Alternatively, we used arrival time distribution (ATD) profiles that were characteristic of individual isomers and were acquired for protonated molecules or fragment ions. Both approaches provided comparable results. Calculations were used to determine the structures and collision cross sections of both protonated and lithiated isomers that accurately characterized their ion mobility properties. The applicability of ATD profiles to isomer differentiation was demonstrated using direct infusion and flow injection analysis with electrospray of solutions, as well as desorption electrospray of solid samples. Data processing was performed by applying multiple linear regression to the ATD profiles. Using the proposed ATD profile-based approach, the relationships between the determined and given content of isomers showed good linearity with coefficients of determination typically greater than 0.99. Flow injection analysis using an autosampler allowed us to rapidly determine isomeric ratios in a sample containing two isomeric pairs with a minor isomer of 10% (determined 9.3% of 3-MMC and 11.0% of 3-FMC in a mixture with buphedrone and 4-FMC). The proposed approach is not only useful for NPS, but also may be applicable to small isomeric molecules analyzed by ion mobility when complete separation of isomers is not achieved.

Wetland technology for the treatment of HCH-contaminated water – Case study at Hajek site

Hexachlorocyclohexanes (HCH) isomers and their transformation products, such as chlorobenzenes (ClB), generate severe and persistent environmental problems at many sites worldwide. The Wetland technology employing oxidation-reduction, biosorption, biodegradation and phytoremediation methods can sufficiently treat HCH-contaminated water. The treatment process is inherently natural and requires no supplementary chemicals or energy. The prototype with a capacity of 3 L/s was installed at Hajek quarry spoil heap (CZ), to optimize the technology on a full scale. The system is fed by drainage water with an average concentration of HCH 129 μg/L, ClB 640 μg/L and chlorophenols (ClPh) of 16 μg/L. The system was tested in two years of operation, regularly monitored for HCH, ClB and ClPh, and maintained to improve its efficiency. The assessment was not only for environmental effects but also for socio and economic indicators. During the operation, the removal efficiency of HCH ranged from 53.5 % to 96.9 % (83.9 % on average) depending on the flow rate. Removal efficiency was not uniform for individual HCH isomers but exhibited the trend: α = γ = δ > β = ε. The improved water quality was reflected in a biodiversity increase expressed by a number of phytobenthos (diatoms) species, a common biomarker of aquatic environment quality. The Wetland outranked the conventional WWTP in 10 out of the 15 general categories, and it is the most relevant scenario from the socio, environmental, and economic aspects.

Anaerobic biotransformation of hexachlorocyclohexane isomers in aqueous condition: Dual C[sbnd]Cl isotope fractionation and impact on microbial community compositions

Hexachlorocyclohexane (HCH) isomers are persistent organic pollutants (POPs) with high toxicity, lipid solubility, chemical stability. Despite the current ban on usage of Lindane, residual contamination cannot be ignored, and HCH are frequently detected in groundwater and threaten human health. Cultures capable of degrading α-HCH, β-HCH, γ-HCH, and δ-HCH individually have been enriched in anoxic aqueous conditions. Compound-Specific Isotope Analysis (CSIA) was applied to examine the transformation mechanisms of different HCH isomers by the four enrichment cultures. 16S rRNA sequencing techniques were employed to examine the community composition of the enrichment cultures and detect changes in these communities resulting from adding individual HCH isomers. The results indicated that the ability of the enrichment cultures for dichloroelimination of HCH isomers was inconsistent. During dichloroelimination, different bond cleavage mode of β- and δ-HCH led to distinct isotopic effects. HCH isomers had significant impact on the microbial community, while different microbial communities showed comparable isotopic effects during the transformation of a specific HCH isomer. In addition, bacteria in the phyla Proteobacteria and Firmicutes were proposed as the dominant dechlorinators. This study provides a novel perspective on the mode of bond cleavage during HCH dichloroelimination and the effect of HCH on microbial communities, which could potentially support the evaluation of HCH transformation by CSIA and their effects on the microecosystems of groundwater.

Prevalence of carboxy-Δ8-tetrahydrocannabiniol in antidoping samples.

Δ9-Tetrahydrocannabinol (Δ9-THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8-tetrahydrocannabinol (Δ8-THC), have become available for sale. Δ8-THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8-THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9-THC (Δ9-THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8-THC, which metabolizes to Δ8-THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8-THC. This study aims to estimate the prevalence of Δ8-THC use in the athlete community by monitoring for Δ8-THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9-THC-COOH. One thousand samples with a presumptive Δ9-THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8-THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

Isomeric and rotational effects in the chemi-ionisation of 1,2-dibromoethene with metastable neon atoms.

The specific geometry of a molecule can have a pronounced influence on its chemical reactivity. However, experimental data on reactions of individual molecular isomers are still sparse because they are often difficult to separate and frequently interconvert into one another under ambient conditions. Here, we employ a novel crossed-beam experiment featuring an electrostatically controlled molecular beam combined with a source for radicals and metastables to spatially separate the cis and trans stereoisomers as well as individual rotational states of 1,2-dibromoethene and study their specific reactivities in the chemi-ionisation reaction with excited neon atoms. The experiments reveal pronounced isomeric and rotational specificities in the rates and product branching ratios of the reaction. The present study underlines the importance and combined role of molecular geometry and of rotational motion in the dynamics of chemi-ionisation reactions.

Infrared spectra of TiO2 clusters for hot Jupiter atmospheres

Context. Clouds appear to be an unavoidable phenomenon in cool and dense environments. Hence, their inclusion is a necessary part of explaining observations of exoplanet atmospheres, most recently those of WASP 96b with the James Webb Space Telescope (JWST). Understanding the formation of cloud condensation nuclei in non-terrestrial environments is therefore crucial in developing accurate models to interpret current and future observations. Aims. The goal of the paper is to support observations with infrared spectra for (TiO2)N clusters to study cloud formation in exoplanet atmospheres. Methods. We derived vibrational frequencies from quantum-chemical calculations for 123 (TiO2)-clusters and their isomers and we evaluated their line-broadening mechanisms. Cluster spectra were calculated for several atmospheric levels for two example exoplanet atmospheres (WASP 121b-like and WASP 96b-like) to identify possible spectral fingerprints for cloud formation. Results. The rotational motion of clusters and the rotational transitions within them cause significant line broadening, so that individual vibrational lines are broadened beyond the spectral resolution of the medium-resolution mode of the JWST mid-infrared instrument (MIRI) at R = 3000. However, each individual cluster isomer exhibits a ‘fingerprint’ IR spectrum. In particular, larger (TiO2) clusters have distinctly different spectra from smaller clusters. The morning and evening terminator for the same planet can exhibit different total absorbances, due to the greater abundance of different cluster sizes. Conclusions. The largest (TiO2) clusters are not necessarily the most abundant (TiO2) clusters in the high-altitude regions of ultra-hot Jupiters and the different cluster isomers do contribute to the local absorbance. Planets with a considerable day-night asymmetry will be most suitable in the search for (TiO2) cluster isomers with the goal of improving cloud formation modelling.

Disparate Redox Potentials in Mixed Isomer Electrolytes Reduce Voltage Efficiency of Energy Dense Flow Batteries

Electrolytes containing multiple redox couples are promising for improving the energy density of flow batteries. Here, two chelated chromium complexes that are structural isomers are characterized and combined to generate electrolytes containing up to 2 M of active species, corresponding to 53.6 Ah L−1. The mixed isomer approach enables a significantly higher active material content than the individual materials would allow, affording energy dense cells with Coulombic efficiencies of ≥99.6% at 100 mA cm−2 and an open circuit voltage of 1.65 V at 50% state-of-charge. This high concentration, however, comes with a caveat; at a given concentration, an equimolar mixed electrolyte leads to lower voltage efficiency compared to using the individual isomers, while Coulombic efficiency remains constant. Our work demonstrates that exploiting structural isomerism is an efficient approach to improve capacity, but active materials must be selected carefully in mixed systems as differences in operating potentials negatively affect energy efficiency.

Measurements of volatile organic compounds in ambient air by gas-chromatography and real-time Vocus PTR-TOF-MS: calibrations, instrument background corrections, and introducing a PTR Data Toolkit

Abstract. Volatile organic compound (VOC) emissions and subsequent oxidation contribute to the formation of secondary pollutants and poor air quality in general. As more VOCs at lower mixing ratios have become the target of air quality investigations, their quantification has been aided by technological advancements in proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS). However, such quantification requires appropriate instrument background measurements and calibrations, particularly for VOCs without calibration standards. This study utilized a Vocus PTR-TOF-MS coupled with a gas chromatograph for real-time and speciated measurements of ambient VOCs in Boulder, Colorado, during spring 2021. The aim of these measurements was to understand and characterize instrument response and temporal variability as to inform the quantification of a broader range of detected VOCs. Fast, frequent calibrations were made every 2 h in addition to daily multipoint calibrations. Sensitivities derived from the fast calibrations were 5 ± 6 % (average and 1 standard deviation) lower than those derived from the multipoint calibrations due to an offset between the calibrations and instrument background measurement. This offset was caused, in part, by incomplete mixing of the standard with diluent. These fast calibrations were used in place of a normalization correction to account for variability in instrument response and accounted for non-constant reactor conditions caused by a gradual obstruction of the sample inlet. One symptom of these non-constant conditions was a trend in fragmentation, although the greatest observed variability was 6 % (1 relative standard deviation) for isoprene. A PTR Data Toolkit (PTR-DT) was developed to assess instrument performance and rapidly estimate the sensitivities of VOCs which could not be directly calibrated on the timescale of the fast calibrations using the measured sensitivities of standards, molecular properties, and simple reaction kinetics. Through this toolkit, the standards' sensitivities were recreated within 1 ± 8 % of the measured values. Three clean-air sources were compared: a hydrocarbon trap, zero-grade air and ultra-high purity nitrogen, and a catalytic zero-air generator. The catalytic zero-air generator yielded the lowest instrument background signals for the majority of ions, followed by the hydrocarbon trap. Depending on the ionization efficiency, product ion fragmentation, ion transmission, and instrument background, standards' limits of detection (5 s measurement integration) derived from the catalytic zero-air generator and the fast calibration sensitivities ranged from 2 ppbv (methanol) to 1 pptv (decamethylcyclopentasiloxane; D5 siloxane) with most standards having detection limits below 20 pptv. Finally, applications of measurements with low detection limits are considered for a few low-signal species including sub-parts-per-trillion by volume (pptv) enhancements of icosanal (and isomers; 1 min average) in a plume of cooking emissions, and sub-parts-per-trillion by volume enhancements in dimethyl disulfide in plumes containing other organosulfur compounds. Additionally, chromatograms of hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, and decamethylcyclopentasiloxane (D3, D4, and D5 siloxanes, respectively), combined with high sensitivity, suggest that online measurements can reasonably be associated with the individual isomers.

Absolute configuration assignment of highly fluorinated carboxylic acids via VCD and MRR spectroscopy

Chiral analysis has become a crucial step in studying the stereospecific synthesis of Active Pharmaceutical Ingredients (APIs). Both Vibrational Circular Dichroism (VCD) and Molecular Rotational Resonance (MRR) spectroscopy are capable of determining absolute configurations (ACs) via comparison of experimental and calculated data. In this regard, each technique has its own caveats. In VCD analysis, accurate prediction of the normal modes as well as rigorous conformational searches of both the analyte and potential (self-)aggregation products are required to optimally match experimental spectra. In MRR analysis, chiral species are resolved through complexation with a chiral tag to prepare spectrally distinct diastereomeric complexes. Although individual complex isomers can be distinguished, spectral assignments need to be matched to unique isomer geometries for unambiguous AC assignment. In this work, the ACs of two highly fluorinated carboxylic acids were successfully assigned using VCD and MRR spectroscopy. In the VCD analysis, the M06-2X functional was demonstrated to be superior to B3LYP and B3LYP-GD3 in accurately predicting the C–F normal modes and both monomeric and dimeric spectral contributions were observed. In a similar analysis with broadband MRR, most experimentally identified geometries had more than one possible computational match. Nevertheless, careful consideration of the chiral tag, as well as additional isomer assignments, resulted in successful assignment of the AC. This comparative study demonstrates the power of contemporary VCD analysis and the unique contributions of MRR to the analytical toolbox.

Safety of a change in specifications of the novel food oleoresin from Haematococcus pluvialis containing astaxanthin pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of a change of specifications of the novel food (NF) oleoresin from Haematococcus pluvialis containing astaxanthin (ATX) pursuant to Regulation (EU) 2015/2283. The NF is already authorised as ingredient for the use in food supplements as defined in Directive 2002/46EC in accordance to Regulation (EU) 2017/2470. The NF concerns an oleoresin which contains ~ 10% ATX, obtained by supercritical CO2 extraction of the homogenised and dried biomass of cultivated H. pluvialis. This NF has been assessed by the Panel in 2014. With the present dossier, the applicant proposed to lower the minimum specification limits for protein and ATX monoesters for the NF, and to increase the maximum specification limit for therelative amount of ATX diesters in total ATX. An increase of the maximum specification limit for the 9-cis isomer is also applied for. Although the data are limited regarding bioavailability and distribution in humans of these three naturally occurring ATX isomers, the available in vitro and in vivo data suggest that the 13-cis rather than the 9-cis ATX is selectively absorbed, i.e. has a higher bioavailability and/or possibly emerges from isomerisation of all-trans ATX. The Panel notes that thetoxicity of the individual ATX isomers has not been studied individually. However, the ADI of 0.2 mg/kg, which was established for synthetic ATX and ATX from H. pluvialis, applies also for ATX in the oleoresin from H. pluvialis with the proposed changes of specifications. The Panel concludes that the NF, oleoresin from H. pluvialis containing ATX, is safe with the proposed specification limits.

Carotenoids and tocols comparison in different Subspecies of Triticum turgidum and aestivum

Carotenoids and Tocols in six genotypes of Triticum turgidum ssp. durum, five of Triticum turgidum ssp. dicoccum, four of Triticum aestivum ssp. aestivum, and six of Triticum aestivum ssp. spelta were investigated. The aim of the present study was to identify, quantify, and compare the content of tocopherols, tocotrienols, and carotenoids in different primitive and modern genotypes of wheat species in order to evaluate the lines with the highest content and possibly use them for selection and breeding programs. The Triticum durum group showed the highest mean content of total carotenoids, with lutein being the most abundant, accounting for 80.12 % (Triticum spelta) to 86.65 % (Triticum turgidum) of total carotenoids. Among the genotypes, Line 6 (Triticum durum) had the highest lutein content (12.17 μg g−1), significantly differing from the lines within its group and the other groups of dicoccum, aestivum, and spelta.Triticum dicoccum exhibited a lower average content of total tocols compared to other Triticum species. The tocols profile showed a prevalence of tocotrienols over tocopherols. β + γ-T3 was the most abundant individual tocol isomer in all Triticum genotypes, contributing for 50.40 % (Triticum ssp. aestivum) and 42.50 % (Triticum spelta) of the total content, respectively. The highest β + γ-T3 content (23.83 μg/g) was found in Line 6 of Triticum durum. Correlation, principal component, and cluster analyses revealed positive correlations between total tocols and β/γ tocotrienols, significant differences between various groups of the same species, formation of six clusters labeled as I to VI, and the identification of genotypes from the same species grouped in different clusters.

Semicarbazones of alkyl 3-nitropyruvates: Synthesis and structural features

Semicarbazones of alkyl 3-nitropyruvates were obtained by the reaction between alkyl 3-bromo-3-nitroacrylates and semicarbazides. The products were formed as E/Z isomer mixtures from which some representatives of the individual isomers were isolated. The possibility of their isomerization was shown.

Countercurrent chromatography separation of vitamin E isomers in a co-current mode.

Vitamin E represents a group of lipophilic phenolic compounds, including α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, and α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol isomers. Different forms of vitamin E have been proven to exhibit varying biological activities. However, due to their structural similarities, the separation of vitamin E isomers is a challenging task. Therefore, it is crucial to establish an efficient method for isolating individual isomers. In this study, co-current countercurrent chromatography was employed to isolate vitamin E isomers from commercial capsules using a n-heptane-methanol-water (10:9.5:0.5, v/v) solvent system. The partition coefficients of the main constituents in the capsules ranged from 0.94 to 6.23, requiring over 450 min for a complete separation. To improve separation efficiency, a co-current elution mode was implemented and the flow rates of the two liquid phases as well as sample amount were examined. The results suggested that increasing the flow rate of the stationary phase and sample size could result in more effective separation, shorter separation time, and higher yield. It proved that co-current countercurrent chromatography was an effective method for the separation of vitamin E isomers.

Mixed Isomer Chromium-Chelates for High Concentration Aqueous Flow Battery Electrolytes

Redox flow batteries have attracted attention for their potential for large-scale energy storage, largely due to their ability to decouple power and energy. However, their limited energy density is often cited as a major drawback preventing adoption at scale. Therefore, there is interest in research into increasing the solubility of the active material in these electrolytes. One such method is active materials with multiple redox events. Promising materials are, however, often poorly soluble, and computational studies showed that increasing differences in redox potential between e.g., two redox events will decrease the energy efficiency of the cell.1 Here, two Cr-chelate structural isomer complexes, with redox potentials 100 mV apart are characterized and compared electrochemically. The two chromium complexes are combined in mixed-anion electrolytes to generate an electrolyte of 2 M active species, corresponding to a capacity of 53.6 Ah L−1, significantly higher than the individual isomers could achieve. At 100 mA cm−2 the cell achieved average Coulombic efficiencies of 99.6% and an equilibrium cell potential of 1.65 V at 50% State-of-Charge. Due to high viscosity of the electrolyte, the voltaic efficiency was only 67%. At a 1 M concentration, the equimolar mixed-anion electrolyte had a comparable current efficiency to the individual Cr-isomers (within 0.2%) but lowered voltaic efficiency (75.8% compared to 77.8% and 78.6%). The lowered voltaic efficiency is attributed to inefficiencies caused by non-concerted multiple redox events. The high cell voltage and increased combined electrolyte capacity demonstrate that employing chirality, chelate geometry, and structural isomers in a mixed-anion approach can significantly increase overall active material concentrations, but that there are consequences for cell performance when using multiple molecules with disparate reduction potentials in an electrolyte. This highlights that concerted transitions should be prioritized in order to capitalize on multi-electron redox species.

Top-Down Protein Analysis by Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry (Tandem-TIMS/MS) Coupled with Ultraviolet Photodissociation (UVPD) and Parallel Accumulation/Serial Fragmentation (PASEF) MS/MS Analysis.

"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their intact mass as well as the observed fragmentation pattern in tandem mass spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry-mass spectrometry (IM/MS) methods have gained traction for top-down experiments, either by allowing top-down analysis of individual isomers or alternatively by improving signal/noise and dynamic range for fragment ion assignment. We recently described the construction of a tandem-trapped ion mobility spectrometer/mass spectrometer (tandem-TIMS/MS) coupled with an ultraviolet (UV) laser and demonstrated a proof-of-principle for top-down analysis by UV photodissociation (UVPD) at 2-3 mbar. The present work builds on this with an exploration of a top-down method that couples tandem-TIMS/MS with UVPD and parallel-accumulation serial fragmentation (PASEF) MS/MS analysis. We first survey types and structures of UVPD-specific fragment ions generated in the 2-3 mbar pressure regime of our instrument. Notably, we observe UVPD-induced fragment ions with multiple conformations that differ from those produced in the absence of UV irradiation. Subsequently, we discuss how MS/MS spectra of top-down fragment ions lend themselves ideally for probability-based scoring methods developed in the bottom-up proteomics field and how the ability to record automated PASEF-MS/MS spectra resolves ambiguities in the assignment of top-down fragment ions. Finally, we describe the coupling of tandem-TIMS/MS workflows with UVPD and PASEF-MS/MS analysis for native top-down protein analysis.

Identification and Imaging of Prostaglandin Isomers Utilizing MS3 Product Ions and Silver Cationization.

Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods.

Isomer-Resolved Mass Spectrometry Imaging of Acidic Phospholipids.

The biological functions of lipids are entirely dependent on their molecular structures with even small changes in structure─such as different sites of unsaturation─providing critical markers for changes in the underlying metabolism. Conventional mass spectrometry imaging (MSI) approaches, however, face the twin challenges of mixture and structural complexity and are typically unable to differentiate lipid isomers that differ only in the position(s) of carbon-carbon double bonds. Recent coupling of ozone-induced dissociation (OzID) with matrix-assisted laser desorption/ionization (MALDI)-MSI has demonstrated the potential to map changes in individual double-bond isomers, thus enabling visualization of the modulation in lipid desaturation in adjacent tissue types. This has, to date, only been performed in positive-ion mode due to a generally higher abundance of phosphatidylcholines (PC) in mammalian tissues and the efficient desorption/ionization of this lipid subclass. Many other glycerophospholipids (GPLs), however, are better detected in negative-ion mode as deprotonated anions. Recently, OzID has been implemented on a traveling-wave ion-mobility mass spectrometer (Waters, SYNAPT G2-Si) that provides a 50-fold increase in the rate of the gas-phase reaction between ionized lipids and ozone and a commensurate increase in sensitivity for isomer-resolved mass spectrometry. These gains are exploited here to interrogate the distributions of anionic GPL isomers in biological tissues, covering the subclasses phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). Exploiting both ozone- and collision-induced dissociation in a single acquisition simultaneously identifies sites of unsaturation and acyl chain composition from the same mass spectrum.

Diastereomer Resolution of M4L6 Coordination Cages by Ultra‐High‐Resolution Ion‐Mobility Mass Spectrometry

AbstractCoordination 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.