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The fragmentation of ion clusters within the accelerating fields of ionic liquid ion sources (ILISs) is well documented and degrades ILIS performance and lifetime. Some of the most popular ILIS liquids, such as EMI-BF4 (1-Ethyl-3-methylimidazolium tetrafluoroborate) and EMI-Im (1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide), emit clusters with lifetimes as low as ∼1 ns. Studies of fragmentation within the accelerating field typically rely on measuring the plume energy distribution averaged over all plume species and comparing those measurements with numerical simulations to estimate ion cluster lifetimes. Here, for the first time, we estimate EMI-BF4 cluster lifetimes by analyzing the energy distributions of individual plume species. We use this novel analysis method to estimate mean lifetimes of positive EMI-BF4 ion clusters from previously published experimental data. We find that the mean lifetime ranges from τ = 3.7 ns to τ = 124 ns for [ EM I + ] [ EMI - B F 4 ] dimers and ranges from τ = 1.5 ns to τ = 23 ns for [ EM I + ] [ EMI - B F 4 ] 2 trimers. Fitting those data to an analytical fragmentation model, we estimate the binding energy and temperature as Δ G S 0 = 0.49 eV and T = 394 K for dimers and Δ G S 0 = 0.40 eV and T = 365 K for trimers. Comparing our results with previous studies supports the conclusion that clusters are emitted with a wide distribution of internal energies, contrary to the common assumption of single internal energy for each species. [ABSTRACT FROM AUTHOR]

Intramolecular vibrational energy redistribution (IVR) has a profound impact on dynamic processes. We have studied two types of IVR processes, restricted and dissipative, and ultrafast dynamics of the S1 state of 2,4-difluoroanisole using time-resolved photoelectron spectroscopy and time-of-flight mass spectroscopy. The restricted IVR occurs in the intermediate regime of 219 cm−1 vibrational level, and the dissipative IVR occurs in the statistical regime of 1200 cm−1. The lifetimes of IVR processes are measured to be 90 and 11 ps, respectively, depending on the internal energies of the S1 state and differ by a factor of eight. Similar subsequent dynamics were observed at two vibrational levels in the S1 state. The population undergoes IVR following the initial excitation and subsequently leaks into a triplet state, accompanied by intersystem crossing within ∼400 ps followed by a slower nonradiative relaxation of the triplet state on the nanosecond time scale. Furthermore, the values of 3s and 3px Rydberg states of 2,4-difluoroanisole were experimentally determined to be 5.02 and 6.28 eV. [ABSTRACT FROM AUTHOR]

The interactions of electrons with molecular systems under various conditions are essential to interdisciplinary research fields extending over the fundamental and applied sciences. In particular, investigating electron-induced ionization and dissociation of molecules may shed light on the radiation damage to living cells, the physicochemical processes in interstellar environments, and reaction mechanisms occurring in combustion or plasma. We have, therefore, studied electron-induced ionization and dissociation of the gas phase 3,4-dihydro-2H-pyran (DHP), a cyclic ether appearing to be a viable moiety for developing efficient clinical pharmacokinetics and revealing the mechanisms of biofuel combustion. The mass spectra in the m/z = 10–90 mass range were measured at several different energies of the ionizing electron beam using mass spectrometry. The mass spectra of DHP at the same energies were simulated using on-the-fly semi-classical molecular dynamics (MD) within the framework of the QCxMS formalism. The MD settings were suitably adjusted until a good agreement with the experimental mass spectra intensities was achieved, thus enabling a reliable assignment of cations and unraveling the plausible fragmentation channels. Based on the measurement of the absolute total ionization cross section of DHP (18.1 ± 0.9) × 10−16 cm2 at 100 eV energy, the absolute total and partial ionization cross sections of DHP were determined in the 5–140 eV electron energy. Moreover, a machine learning algorithm that was trained with measured cross sections from 25 different molecules was used to predict the total ionization cross section for DHP. Comparison of the machine learning simulation with the measured data showed acceptable agreement, similar to that achieved in past predictions of the algorithm. [ABSTRACT FROM AUTHOR]

Stable isotope analysis of bone provides insight into animal foraging and allows for ecological reconstructions over time, however pre-treatment is required to isolate collagen. Pre-treatments typically consist of demineralization to remove inorganic components and/or lipid extraction to remove fats, but these protocols can differentially affect stable carbon (δ13C) and nitrogen (δ15N) isotope values depending on the chemicals, tissues, and/or species involved. Species-specific methodologies create a standard for comparability across studies and enhance understanding of collagen isolation from modern cetacean bone. Elemental analyzers coupled to isotope ratio mass spectrometers were used to measure the δ13C and δ15N values of powdered killer whale (Orcinus orca) bone that was intact (control) or subjected to one of three experimental conditions: demineralized, lipid-extracted, and both demineralized and lipid-extracted. Additionally, C:N ratios were evaluated as a proxy for collagen purity. Lastly, correlations were examined between control C:N ratios vs. historical age and control C:N ratios vs. sample characteristics. No significant differences in the δ15N values were observed for any of the experimental protocols. However, the δ13C values were significantly increased by all three experimental protocols: demineralization, lipid extraction, and both treatments combined. The most influential protocol was both demineralization and lipid extraction. Measures of the C:N ratios were also significantly lowered by demineralization and both treatments combined, indicating the material was closer to pure collagen after the treatments. Collagen purity as indicated via C:N ratio was not correlated with historical age nor sample characteristics. If only the δ15N values from killer whale bone are of interest for analysis, no pre-treatment seems necessary. If the δ13C values are of interest, samples should be both demineralized and lipid-extracted. As historical age and specimen characteristics are not correlated with sample contamination, all samples can be treated equally.

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.

We calculate electron impact partial and total ionization cross sections of R-carvone (C10H14O), 2-butanol (C4H10O), imidazole (C3H4N2), and 2-nitroimidazole (C3H3N3O2). We have used the Binary Encounter Bethe (BEB) model to obtain total electron impact ionization cross sections (TICSs). The modified BEB method in combination with mass spectrum data of the molecules is used to calculate the partial ionization cross section (PICS) of the cationic fragments dissociating from the parent molecule. Our PICS data for R-carvone and 2-butanol are in good agreement with the experimental data for all the cation fragments along with the TICS data. For imidazole and 2-nitroimidazole, the estimates of the PICS are reported for the first time in the present study. We have found that both the modified BEB method and the mass spectrum dependence method work effectively to estimate PICS if we have information about the appearance energies and relative abundance data of the target under investigation. [ABSTRACT FROM AUTHOR]

Caffeine is an ergogenic substance that is consumed globally in many forms. The use of buccally absorbable formulations instead of gastrointestinal uptake has become increasingly popular over the years, especially when accelerated absorption with minimal gastrointestinal stress is desired. This study investigated the impact of five different formulations and administration routes of caffeine on the whole blood concentrations of caffeine, paraxanthine, and theobromine: caffeinated capsules, tablets, shots, pouches, and chewing gums. A uniform dose of caffeine (200 mg) was administered to 16 healthy recreational athletes (26.0 ± 2.1 years) using a randomized crossover design. Samples were taken in the form of dried blood spots at 16 different time points in a 2-hr timeframe after drug administration. The samples were analyzed using a validated liquid chromatography–tandem mass spectrometry method. The results for caffeine showed no significant differences in the overall bioavailability (area under the concentration–time curve), maximal concentration, and time to maximum concentration. However, when analyzing the bioavailability of caffeine in the first 5, 10, and 15 min, the liquid caffeine formulation was superior to other administered forms (p <.05). This indicates that caffeine solubility has a major influence on its absorption rate. In sports, the rate of caffeine absorption must be considered, not only when ingesting anhydrous caffeine, but also when choosing buccal absorption. These findings imply that general guidelines for ergogenic caffeine use should consider the formulation used and, accordingly, the corresponding route of absorption. [ABSTRACT FROM AUTHOR]

In the present work, we report an update and extension of the previous ion-pair formation study of Hubers, M.M.; Los, J. Chem. Phys.1975, 10, 235-259, noting new fragment anions from time-of-flight mass spectrometry. The branching ratios obtained from the negative ions formed in K + SF6 collisions, in a wide energy range from 10.7 up to 213.1 eV in the centre-of-mass frame, show that the main anion is assigned to SF5- and contributing to more than 70% of the total ion yield, followed by the non-dissociated parent anion SF6- and F-. Other less intense anions amounting to

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

Protein-protein interactions (PPIs) are at the heart of the molecular landscape permeating life. Proteomics studies can explore this protein interaction landscape using mass spectrometry (MS). Thanks to their high sensitivity, mass spectrometers can easily identify thousands of proteins within a single sample, but that same sensitivity generates tangled spiderwebs of data that hide biologically relevant findings. So, what does a researcher do when she finds herself walking into spiderwebs? In a field focused on discovery, MS data require rigor in their analysis, experimental validation, or a combination of both. In this Review, we provide a brief primer on MS-based experimental methods to identify PPIs. We discuss approaches to analyze the resulting data and remove the proteomic background. We consider the advantages between comprehensive and targeted studies. We also discuss how scoring might be improved through AI-based protein structure information. Women have been essential to the development of proteomics, so we will specifically highlight work by women that has made this field thrive in recent years.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

In the modern "omics" era, measurement of the human exposome is a critical missing link between genetic drivers and disease outcomes. High-resolution mass spectrometry (HRMS), routinely used in proteomics and metabolomics, has emerged as a leading technology to broadly profile chemical exposure agents and related biomolecules for accurate mass measurement, high sensitivity, rapid data acquisition, and increased resolution of chemical space. Non-targeted approaches are increasingly accessible, supporting a shift from conventional hypothesis-driven, quantitation-centric targeted analyses toward data-driven, hypothesis-generating chemical exposome-wide profiling. However, HRMS-based exposomics encounters unique challenges. New analytical and computational infrastructures are needed to expand the analysis coverage through streamlined, scalable, and harmonized workflows and data pipelines that permit longitudinal chemical exposome tracking, retrospective validation, and multi-omics integration for meaningful health-oriented inferences. In this article, we survey the literature on state-of-the-art HRMS-based technologies, review current analytical workflows and informatic pipelines, and provide an up-to-date reference on exposomic approaches for chemists, toxicologists, epidemiologists, care providers, and stakeholders in health sciences and medicine. We propose efforts to benchmark fit-for-purpose platforms for expanding coverage of chemical space, including gas/liquid chromatography-HRMS (GC-HRMS and LC-HRMS), and discuss opportunities, challenges, and strategies to advance the burgeoning field of the exposome.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

Posttraumatic stress disorder (PTSD) can develop after trauma exposure. Some studies report that women develop PTSD at twice the rate of men, despite greater trauma exposure in men. Lipids and their metabolites (lipidome) regulate a myriad of key biological processes and pathways such as membrane integrity, oxidative stress, and neuroinflammation in the brain by maintaining neuronal connectivity and homeostasis. In this study, we analyzed the lipidome of 40 adults with PTSD and 40 trauma-exposed non-PTSD individuals (n = 20/sex/condition; 19-39 years old). Plasma samples were analyzed for lipidomics using Quadrupole Time-of-Flight (QToF) mass spectrometry. Additionally, ~ 90 measures were collected, on sleep, and mental and physical health indices. Poorer sleep quality was associated with greater PTSD severity in both sexes. The lipidomics analysis identified a total of 348 quantifiable known lipid metabolites and 1951 lipid metabolites that are yet unknown; known metabolites were part of 13 lipid subclasses. After adjusting for BMI and sleep quality, in women with PTSD, only one lipid subclass, phosphatidylethanolamine (PE) was altered, whereas, in men with PTSD, 9 out of 13 subclasses were altered compared to non-PTSD women and men, respectively. Severe PTSD was associated with 22% and 5% of altered lipid metabolites in men and women, respectively. Of the changed metabolites, only 0.5% measures (2 PEs and cholesterol) were common between women and men with PTSD. Several sphingomyelins, PEs, ceramides, and triglycerides were increased in men with severe PTSD. The correlations between triglycerides and ceramide metabolites with cholesterol metabolites and systolic blood pressure were dependent upon sex and PTSD status. Alterations in triglycerides and ceramides are linked with cardiac health and metabolic function in humans. Thus, disturbed sleep and higher body mass may have contributed to changes in the lipidome found in PTSD.

Electronic cigarette smoking (or vaping) is on the rise, presenting questions about the effects of secondhand exposure. The chemical composition of vape emissions was examined in the exhaled breath of eight human volunteers with the high chemical specificity of complementary online and offline techniques. Our study is the first to take multiple exhaled puff measurements from human participants and compare volatile organic compound (VOC) concentrations between two commonly used methods, proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS) and gas chromatography (GC). Five flavor profile groups were selected for this study, but flavor compounds were not observed as the main contributors to the PTR-ToF-MS signal. Instead, the PTR-ToF-MS mass spectra were overwhelmed by e-liquid thermal decomposition and fragmentation products, which masked other observations regarding flavorings and other potentially toxic species associated with secondhand vape exposure. Compared to the PTR-ToF-MS, GC measurements reported significantly different VOC concentrations, usually below those from PTR-ToF-MS. Consequently, PTR-ToF-MS mass spectra should be interpreted with caution when reporting quantitative results in vaping studies, such as doses of inhaled VOCs. Nevertheless, the online PTR-ToF-MS analysis can provide valuable qualitative information by comparing relative VOCs in back-to-back trials. For example, by comparing the mass spectra of exhaled air with those of direct puffs, we can conclude that harmful VOCs present in the vape emissions are largely absorbed by the participants, including large fractions of nicotine.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Purines are the building blocks of DNA/RNA, energy substrates, and cofactors. Purine metabolites, including ATP, GTP, NADH, and coenzyme A, are essential molecules in diverse biological processes such as energy metabolism, signal transduction, and enzyme activity. When purine levels increase, excess purines are either recycled to synthesize purine metabolites or catabolized to the end product uric acid. Purine catabolism increases during states of low oxygen tension (hypoxia and ischemia), but this metabolic pathway is incompletely understood in the context of histotoxic hypoxia (i.e., inhibition of oxygen utilization despite normal oxygen tension). In rabbits exposed to cyanide-a classical histotoxic hypoxia agent-we demonstrated significant increases in several concordant metabolites in the purine catabolic pathway (including plasma levels of uric acid, xanthosine, xanthine, hypoxanthine, and inosine) via mass spectrometry-based metabolite profiling. Pharmacological inhibition of the purine catabolic pathway with oxypurinol mitigated the deleterious effects of cyanide on skeletal muscle cytochrome c oxidase redox state, measured by non-invasive diffuse optical spectroscopy. Finally, plasma uric acid levels correlated strongly with those of lactic acid, an established clinical biomarker of cyanide exposure, in addition to a tissue biomarker of cyanide exposure (skeletal muscle cytochrome c oxidase redox state). Cumulatively, these findings not only shed light on the in vivo role(s) of cyanide but also have implications in the field of medical countermeasure (MCM) development.

Epoxyeicosatrienoic acids (EpETrEs) are bioactive lipid mediators of arachidonic acid cytochrome P450 oxidation. In vivo, the free (unbound) form of EpETrEs regulate multiple processes including blood flow, angiogenesis and inflammation resolution. Free EpETrEs are thought to rapidly degrade via soluble epoxide hydrolase (sEH); yet, in many tissues, the majority of EpETrEs are esterified to complex lipids (e.g. phospholipids) suggesting that esterification may play a major role in regulating free, bioactive EpETrE levels. This hypothesis was tested by quantifying the metabolism of intraperitoneally injected free d11-11(12)-Epoxyeicosatrienoic acid (d11-11(12)-EpETrE) in male and female rats. Plasma and tissues (liver, adipose and brain) were obtained 3 to 4 min later and assayed for d11-11(12)-EpETrE and its sEH metabolite, d11-11,12-dihydroxyeicosatrienoic acid (d11-11,12-diHETrE) in both the free and esterified lipid fractions. In both males and females, the majority of injected tracer was recovered in liver followed by plasma and adipose. No tracer was detected in the brain, indicating that brain levels are maintained by endogenous synthesis from precursor fatty acids. In plasma, liver, and adipose, the majority (>54 %) of d11-11(12)-EpETrE was found esterified to phospholipids or neutral lipids (triglycerides and cholesteryl esters). sEH-derived d11-11,12-diHETrE was not detected in plasma or tissues, suggesting negligible conversion within the 3-4 min period post tracer injection. This study shows that esterification is the main pathway regulating free 11(12)-EpETrE levels in vivo.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

BACKGROUND: With the availability of disease-modifying therapies for Alzheimers disease (AD), it is important for clinicians to have tests to aid in AD diagnosis, especially when the presence of amyloid pathology is a criterion for receiving treatment. METHODS: High-throughput, mass spectrometry-based assays were used to measure %p-tau217 and amyloid beta (Aβ)42/40 ratio in blood samples from 583 individuals with suspected AD (53% positron emission tomography [PET] positive by Centiloid > 25). An algorithm (PrecivityAD2 test) was developed using these plasma biomarkers to identify brain amyloidosis by PET. RESULTS: The area under the receiver operating characteristic curve (AUC-ROC) for %p-tau217 (0.94) was statistically significantly higher than that for p-tau217 concentration (0.91). The AUC-ROC for the PrecivityAD2 test output, the Amyloid Probability Score 2, was 0.94, yielding 88% agreement with amyloid PET. Diagnostic performance of the APS2 was similar by ethnicity, sex, age, and apoE4 status. DISCUSSION: The PrecivityAD2 blood test showed strong clinical validity, with excellent agreement with brain amyloidosis by PET.

The purpose of this study was to investigate changes in the lipidome of patients with sepsis to identify signaling lipids associated with poor outcomes that could be linked to future therapies. Adult patients with sepsis were enrolled within 24h of sepsis recognition. Patients meeting Sepsis-3 criteria were enrolled from the emergency department or intensive care unit and blood samples were obtained. Clinical data were collected and outcomes of rapid recovery, chronic critical illness (CCI), or early death were adjudicated by clinicians. Lipidomic analysis was performed on two platforms, the Sciex™ 5500 device to perform a lipidomic screen of 1450 lipid species and a targeted signaling lipid panel using liquid-chromatography tandem mass spectrometry. For the lipidomic screen, there were 274 patients with sepsis: 192 with rapid recovery, 47 with CCI, and 35 with early deaths. CCI and early death patients were grouped together for analysis. Fatty acid (FA) 12:0 was decreased in CCI/early death, whereas FA 17:0 and 20:1 were elevated in CCI/early death, compared to rapid recovery patients. For the signaling lipid panel analysis, there were 262 patients with sepsis: 189 with rapid recovery, 45 with CCI, and 28 with early death. Pro-inflammatory signaling lipids from ω-6 poly-unsaturated fatty acids (PUFAs), including 15-hydroxyeicosatetraenoic (HETE), 12-HETE, and 11-HETE (oxidation products of arachidonic acid [AA]) were elevated in CCI/early death patients compared to rapid recovery. The pro-resolving lipid mediator from ω-3 PUFAs, 14(S)-hydroxy docosahexaenoic acid (14S-HDHA), was also elevated in CCI/early death compared to rapid recovery. Signaling lipids of the AA pathway were elevated in poor-outcome patients with sepsis and may serve as targets for future therapies.

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

This month's column is Part 2 of a contribution from my daughter Glenna, who recently completed her PhD studies in Environmental Science from the University of Copenhagen in Denmark. Her article explores the current landscape of global critical raw materials (CRM) trends in research and the applications of atomic spectroscopy (AS), including inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), and X-ray analytical techniques in their identification of diverse industrial and environmental media, which have been essential in method validation and quantification of CRMs in complex matrices presenting high risks of interference. Some important examples to be presented include rare earth elements (REEs) in water leaching purification (WLP) residues that co-occur with radioactive materials; REEs and other metals in acid mine drainage (AMD) environments; REEs in coal combustion (fly ash) residues; arsenic (As) from groundwater treatment sediment; and platinum-group elements (PGEs) from sewage sludge. The article also classifies the different techniques in use at each stage of the CRM recovery train, investigates present challenges to each analytical method, and discusses the problem-solving tools used., - Rob Thomas, Column Editor Part 1 of this article focused on the overview of critical raw materials (CRMs), the global context for sustainable sourcing of CRMs, and the recent […]

Phosphorus (P) doping in ZnTe grown by molecular beam epitaxy (MBE) under alternating source supply method was investigated to achieve p-type P-doped ZnTe (ZnTe:P) thin films using InP as a P dopant source, and the result was compared with those grown under a simultaneous MBE growth where the source beams were supplied simultaneously. As a result, P concentration in ZnTe thin films was found to increase with increasing the InP flux, and high P concentration up to 6.6 × 1019 cm−3 was confirmed by secondary ion mass spectroscopy (SIMS) analyses. However, In incorporation was also observed in the ZnTe:P thin films, despite that the detected In concentration by SIMS was more than one order of magnitude lower than the P concentration and almost two order of magnitude lower than those grown by the simultaneous MBE. Photoluminescence measurement of ZnTe:P thin film grown under alternating source supply showed a P-related acceptor bound exciton (Ia) peak at 2.37 eV, and the intensity of Ia emission increased after the annealing treatment, indicating the activation of P acceptor. The annealing also decreases the resistivity of the film. The results clearly indicate that the alternating source supply growth is effective to obtain ZnTe:P thin films with better P doping properties. [ABSTRACT FROM AUTHOR]

The role of ambient oxygen gas (O2) on molecular and nanoparticle formation and agglomeration was studied in laser ablation plumes. As a lab-scale surrogate to a high explosion detonation event, nanosecond laser ablation of an aluminum alloy (AA6061) target was performed in atmospheric pressure conditions. Optical emission spectroscopy and two mass spectrometry techniques were used to monitor the early to late stages of plasma generation to track the evolution of atoms, molecules, clusters, nanoparticles, and agglomerates. The experiments were performed under atmospheric pressure air, atmospheric pressure nitrogen, and 20% and 5% O2 (balance N2), the latter specifically with in situ mass spectrometry. Electron microscopy was performed ex situ to identify crystal structure and elemental distributions in individual nanoparticles. We find that the presence of ≈20% O2 leads to strong AlO emission, whereas in a flowing N2 environment (with trace O2), AlN and strong, unreacted Al emissions are present. In situ mass spectrometry reveals that as O2 availability increases, Al oxide cluster size increases. Nanoparticle agglomerates formed in air are found to be larger than those formed under N2 gas. High-resolution transmission electron microscopy demonstrates that Al2O3 and AlN nanoparticle agglomerates are formed in both environments; indicating that the presence of trace O2 can lead to Al2O3 nanoparticle formation. The present results highlight that the availability of O2 in the ambient gas significantly impacts spectral signatures, cluster size, and nanoparticle agglomeration behavior. These results are relevant to understanding debris formation in an explosion event, and interpreting data from forensic investigations. [ABSTRACT FROM AUTHOR]

Site-selective probing of iodine 4d orbitals at 13.1 nm was used to characterize the photolysis of CH2I2 and CH2BrI initiated at 202.5 nm. Time-dependent fragment ion momenta were recorded using Coulomb explosion imaging mass spectrometry and used to determine the structural dynamics of the dissociating molecules. Correlations between these fragment momenta, as well as the onset times of electron transfer reactions between them, indicate that each molecule can undergo neutral three-body photolysis. For CH2I2, the structural evolution of the neutral molecule was simultaneously characterized along the C–I and I–C–I coordinates, demonstrating the sensitivity of these measurements to nuclear motion along multiple degrees of freedom. [ABSTRACT FROM AUTHOR]

We present a surface sampling method for the chemical analysis of liquid-immersed solid samples. Liquid immersion allows samples to be kept in a hydrated state. We employed cavitation generated by high-intensity focused ultrasound for localized material removal. The appropriate transducer–sample distance was determined using the actuating ultrasound transducer prior to sampling, allowing sonication in focus despite surface height variation. We demonstrate the proposed surface sampling method on water-submerged glass cover slides painted with permanent markers, achieving sampling with tunable spot size down to 500 μ m. The removed and collected material was transferred for chemical analysis by electrospray ionization mass spectrometry, which showed mass peaks characteristic to the permanent markers. [ABSTRACT FROM AUTHOR]

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

BACKGROUND: Lipoprotein(a) is a risk factor for cardiovascular events and modifies the benefit of PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors. Lipoprotein(a) concentration can be measured with immunoassays reporting mass or molar concentration or a reference measurement system using mass spectrometry. Whether the relationships between lipoprotein(a) concentrations and cardiovascular events in a high-risk cohort differ across lipoprotein(a) methods is unknown. We compared the prognostic and predictive value of these types of lipoprotein(a) tests for major adverse cardiovascular events (MACE). METHODS: The ODYSSEY OUTCOMES trial (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) compared the PCSK9 inhibitor alirocumab with placebo in patients with recent acute coronary syndrome. We compared risk of a MACE in the placebo group and MACE risk reduction with alirocumab according to baseline lipoprotein(a) concentration measured by Siemens N-latex nephelometric immunoassay (IA-mass; mg/dL), Roche Tina-Quant turbidimetric immunoassay (IA-molar; nmol/L), and a noncommercial mass spectrometry-based test (MS; nmol/L). Lipoprotein(a) values were transformed into percentiles for comparative modeling. Natural cubic splines estimated continuous relationships between baseline lipoprotein(a) and outcomes in each treatment group. Event rates were also determined across baseline lipoprotein(a) quartiles defined by each assay. RESULTS: Among 11 970 trial participants with results from all 3 tests, baseline median (Q1, Q3) lipoprotein(a) concentrations were 21.8 (6.9, 60.0) mg/dL, 45.0 (13.2, 153.8) nmol/L, and 42.2 (14.3, 143.1) nmol/L for IA-mass, IA-molar, and MS, respectively. The strongest correlation was between IA-molar and MS (r=0.990), with nominally weaker correlations between IA-mass and MS (r=0.967) and IA-mass and IA-molar (r=0.972). Relationships of lipoprotein(a) with MACE risk in the placebo group were nearly identical with each test, with estimated cumulative incidences differing by ≤0.4% across lipoprotein(a) percentiles, and all were incrementally prognostic after accounting for low-density lipoprotein cholesterol levels (all spline P≤0.0003). Predicted alirocumab treatment effects were also nearly identical for each of the 3 tests, with estimated treatment hazard ratios differing by ≤0.07 between tests across percentiles and nominally less relative risk reduction by alirocumab at lower percentiles for all 3 tests. Absolute risk reduction with alirocumab increased with increasing lipoprotein(a) measured by each test, with significant linear trends across quartiles. CONCLUSIONS: In patients with recent acute coronary syndrome, 3 lipoprotein(a) tests were similarly prognostic for MACE in the placebo group and predictive of MACE reductions with alirocumab at the cohort level. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01663402.

Small extracellular vesicles (sEVs) are cell-released vesicles ranging from 30-150nm in size. They have garnered increasing attention because of their potential for both the diagnosis and treatment of disease. The diversity of sEVs derives from their biological composition and cargo content. Currently, the isolation of sEV subpopulations is primarily based on bio-physical and affinity-based approaches. Since a standardized definition for sEV subpopulations is yet to be fully established, it is important to further investigate the correlation between the biomolecular composition of sEVs and their physical properties. In this study, we employed a platform combining single-vesicle surface-enhanced Raman spectroscopy (SERS) and machine learning to examine individual sEVs isolated by size-exclusion chromatography (SEC). The biomolecular composition of each vesicle examined was reflected by its corresponding SERS spectral features (biomolecular "fingerprints"), with their roots in the composition of their collective Raman-active bonds. Origins of the SERS spectral features were validated through a comparative analysis between SERS and mass spectrometry (MS). SERS fingerprinting of individual vesicles was effective in overcoming the challenges posed by EV population averaging, allowing for the possibility of analyzing the variations in biomolecular composition between the vesicles of similar and/or different sizes. Using this approach, we uncovered that each of the size-based fractions of sEVs contained particles with predominantly similar SERS spectral features. Indeed, more than 84% of the vesicles residing within a particular group were clearly distinguishable from that of the other EV sub-populations, despite some spectral variations within each sub-population. Our results suggest the possibility that size-based EV fractionation methods produce samples where similarly eluted sEVs are correlated with their respective biochemical contents, as reflected by their SERS spectra. Our findings therefore highlight the possibility that the biogenesis and respective biological functionalities of the various sEV fractions may be inherently different.

Proper neurological function relies on the cellular and molecular microenvironment of the brain, with perturbations of this environment leading to neurological disorders. However, studying the microenvironments of neurological tissue has proven difficult because of its inherent complexity. Both the cell type and metabolomic underpinnings of the cell have crucial functional roles, thus making multimodal characterization methods key to acquiring a holistic view of the brains microenvironment. This study investigates methods for combining matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and immunofluorescence (IF) microscopy to enable the concurrent investigation of cell types and lipid profiles on the same sample. In brief, 1,5-diaminonaphthalene (DAN), α-cyano-4-hydroxy-cinnamic acid (CHCA), and 2,5-dihydroxybenzoic acid (DHB) were tested in addition to instrument-specific parameters for compatibility with IF. Alternatively, the effects of IF protocols on MALDI MSI were also tested, showing significant signal loss with all tested permutations. Ultimately, the use of CHCA for MALDI MSI resulted in the best IF images, while the use of DAN gave the lowest quality IF images. Overall, increasing the laser power and number of shots per laser burst resulted in the most tissue ablation. However, optimized parameter settings allowed for minimal tissue ablation while maintaining sufficient MALDI MSI signal.

In the mirabegron (MIR) synthesis, the N‐nitroso mirabegron (NNM) is obtained during synthetic process of MIR; water is being used in reaction under acidic condition. Nitrite source is from water, and secondary amine source is from MIR as it has secondary amine; NNM is generated as an impurity during the synthesis of MIR. The presence of NNM in MIR could potentially affect its effectiveness. The purpose of this study was to establish a Ultra‐performance liquid chromatography‐mass spectrometry/mass spectrometry (UPLC‐MS/MS) methodology to identify NNM in MIR samples. The method for NNM analysis was developed on Acquity HSS T3 (100*2.1) mm 1.8 μm column with gradient elution using mobile phase consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B). Mass spectrometer with electrospray ionization operated in the MRM mode was used in the analysis of NNM (m/ z 426.20 → 170.00). The UPLC‐MS/MS methodology proposed showed a good linearity (0.02 to 0.72 ppm), good system precision (RSD = 0.57%), good method precision (RSD = 0.87%), acceptable accuracy (94.5–116.5%), low detection limit (0.006 ppm) and low quantification limit (0.02 ppm) for NNM. The UPLC‐MS/MS methodology proposed can be utilized to assess the quality of MIR sample for the presence of NNM impurity. [ABSTRACT FROM AUTHOR]

Rationale: The oxygen stable isotope ratio (δ18O) of the sugar‐rich fraction of fruit juice is important as a tracer of the geographical origin of raw material. This study sought to minimize the inter‐day variation of δ18O attributable to the influence of water to accurately monitor geographical origin labeling. Methods: Two drying devices (freeze dryer and vacuum oven) were compared. Then, two humidity levels (normal and low humidity) at which the samples were placed after drying were compared. The low‐humidity environment was constructed using a glove bag and pure argon gas. δ18O was measured using thermal conversion elemental analyzer/isotope ratio mass spectrometry. Improvements were made to the measurement method based on aforementioned analyses results, and the performance of the initial and improved methods was compared. Results: δ18O of juice dried in a vacuum oven was 3.30‰ lower than that of juice dried in a freeze dryer. Moreover, δ18O of juice samples exposed to normal humidity was 3.74‰ lower than that of samples exposed to low humidity. The combined inter‐day and intra‐day standard deviation was reduced from 1.20‰ in the initial method to 0.42‰ in the improved method. Conclusions: This study describes a pretreatment method for δ18O measurement in the sugar‐rich fraction of fruit juice with less inter‐day variation, and it will be useful for monitoring geographical origin labeling. [ABSTRACT FROM AUTHOR]

Rationale: The analysis of the three sulfur stable isotope ratios (33S/32S, 34S/32S, 36S/32S) is routinely performed by gas‐source isotope ratio mass spectrometry (IRMS) on the SF6 gaseous molecule, collecting SF5+ ions at m/z ~ 127, 128, 129 and 131. High precision and accuracy are commonly achieved owing to a lack of correction because fluorine has only one isotope and the inert nature of the SF6 molecule. The analysis of the 36S/32S ratio is, however, complicated by the low abundance of 36S (~0.015%) and the possible occurrence of trace amounts of fluorocarbon compounds leading to 12C3F5+ ions at m/z ~ 131, i.e. where 36SF5+ ions are collected. Methods: We used gas‐source high‐resolution IRMS to better characterize the nature of possible interferences, and we tested novel types of filaments in order to investigate their influence on possible interferences. Results: We confirm that the 12C3F5+ ion represents the main isobaric interference at m/z ~ 131. We also demonstrate that tungsten fluoride adducts are formed from the reaction of fluorine ions derived during fragmentation of the SF6 molecule with the hot tungsten filament. These reactions lead to the formation of e.g. WF5+, WF4+, WF3+ ions, including doubly charged ions. WF4++, in particular, leads to isobaric interference on m/z ~ 128, 129 and 131 from 180WF4++, 182WF4++ and 186 WF4++ ions, respectively. Because 180W (0.12%) is at low abundance, its influence on δ33S measurements would remain negligible, but 182W (26.5%) and 186W (28.4%) lead to scale contraction for both δ34S and δ36S. Conclusions: Rather than correcting for these interferences, or working at high mass resolution, we suggest avoiding W isobaric interferences by using other types of filaments, with initial reports on both pure Re filaments and Y2O3‐coated W filaments. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Poly‐NHC‐based organometallic assemblies 3‐PF6, 3‐SbF6 and 3‐OTf were obtained and verified by NMR spectroscopy, ESI mass spectrometry and single‐crystal X‐ray diffraction analyses. Controllable structural interconversion was observed between the poly‐NHC‐based organometallic assemblies and their self‐aggregated dimers in solution affected by concentration, solvent and metal ion. 1H NMR spectra of assembly 3‐PF6 in CD3CN at different concentrations demonstrated controllable structural interconversion, and 19F NMR spectrum of assembly 3‐PF6 at high concentration further evidenced the presence of the free hexafluorophosphate anion and encapsulated hexafluorophosphate anion for its two sets of signals. In addition, single‐crystal X‐ray diffraction analysis provided clear evidence that in the solid state, two assemblies 3‐PF6 were vertically stuck, forming a self‐aggregated dimer with an encapsulated hexafluorophosphate anion. Investigating the reversible structural interconversion is beneficial for revealing the intrinsic nature on the atom level and paving the way to design the stimuli‐responsive functional system. [ABSTRACT FROM AUTHOR]

A capillary zone electrophoresis (CZE) system was coupled to an Orbitrap mass spectrometer operating in a data-independent acquisition (DIA) mode for in-depth proteomics analysis. The performance of this CZE-DIA-MS system was systemically evaluated and optimized under different operating conditions. The performance of the fully optimized CZE-DIA-MS system was subsequently compared to the one by using the same CZE-MS system operating in a data-dependent acquisition (DDA) mode. The experimental results show that the numbers of identified peptides and proteins acquired in the DIA mode are much higher than the ones acquired in the DDA mode, especially with the small sample loading amount. Specifically, the numbers of identified peptides and proteins acquired in the DIA mode are 1.8-fold and 2-fold higher than the ones acquired in the DDA mode by using 12.5 ng Hela digests. The proteins identified in the DIA mode also cover almost all the proteins identified in the DDA mode. In addition, a potential cancer biomarker protein, carbohydrate antigen 125, undetected in the DDA mode, can be easily identified in the DIA mode even with 12.5 ng Hela digests. The performance of the CZE-DIA-MS system for in-depth proteomics analysis with a limited sample amount has been fully demonstrated for the first time through this study. [ABSTRACT FROM AUTHOR]

The use of pesticides is often regarded as a fundamental aspect of conventional agriculture. However, these compounds have gained recognition as some of the oldest and most widely employed xenobiotic contaminants, necessitating effective strategies for human biomonitoring. In this context, a method was developed for the determination of 16 legacy organochlorine pesticides, 6 metabolites of current pesticides (2,4-D, malathion, parathion, fipronil, pyraclostrobin, cypermethrin, permethrin, cyfluthrin), and 1 triazine herbicide (atrazine) in serum. Samples were prepared with water, formic acid, acetonitrile, and ultrasound irradiation, followed by solid-phase extraction with Oasis Prime HLB. Subsequently, metabolites from current pesticides underwent derivatization using MTBSTFA with 1% TBDMSCl for analysis via gas chromatography-tandem mass spectrometry (GC–MS/MS), employing an SLB-5MS fused silica capillary column. Analytical curves were generated with limits of quantification from 0.3 to 4.0 ng.mL−1. Accuracy ranged from 69 to 124%, and the coefficient of variation from 2 to 28%. Moreover, determining 1-(4-chlorophenyl)-1H-pyrazol-3-ol was suggested as a biomarker for pyraclostrobin biomonitoring. This analytical approach facilitated the determination of both legacy and metabolites of current pesticides in the same serum sample, presenting an interesting and cost-effective option for large cohorts, and multi-omics studies that evaluate time-dependent biomarkers in blood samples, thereby enabling biomonitoring within the same matrix. Furthermore, a proof-of-concept involving 10 volunteers demonstrated exposure to 9 pesticides at mean concentrations measured in ng mL−1, consistent with findings from various biomonitoring initiatives. [ABSTRACT FROM AUTHOR]

The request for novel hyphenated instruments and techniques, capable of affording exhaustive information and results, is a focus continuously watched out. In this context, the present work aimed at the development of an integrated system combining gas chromatographic (GC) separation with mass spectrometry (MS) and (solid deposition) Fourier transform infrared spectroscopy (FTIR) detection. An external transfer line was designed in the lab for the parallel coupling of the two detectors, in such a way to obtain complementary analytical information consisting of an MS spectrum, an IR spectrum and linear retention indices (LRI), within a single analysis. The instrument performance was demonstrated for the analysis of a commercial mixture consisting of 139 hydrocarbons, comprising linear, branched, unsaturated and aromatic compounds. A 100-m poly(dimethylsiloxane) column was employed for the separation, and the outlet flow was split 95:5 between the IR and MS detectors using two uncoated capillaries. The IR spectra were acquired from solid deposits on a zinc selenide disc (−90 °C), over a spot (detector area) of about 0.1 mm2, in the range of 4000–700 cm−1 and at a resolution of 4 cm−1. Final identification of the separated compounds by a library search was achieved by excluding incorrect results, sequentially using a three-filter approach (85% similarity against reference MS and IR library spectra and ±10 LRI unit tolerance). Based on these preliminary results, the GC-MS/sd-FTIR system is a promising tool for the characterization of complex matrix constituents, for which identification is cumbersome, by using only one detection technique. [ABSTRACT FROM AUTHOR]

Understanding the relationship between the concentration of a drug and its therapeutic efficacy or side effects is crucial in drug development, especially to understand therapeutic efficacy in central nervous system drug, quantifying drug-induced site-specific changes in the levels of endogenous metabolites, such as neurotransmitters. In recent times, evaluation of quantitative distribution of drugs and endogenous metabolites using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) has attracted much attention in drug discovery research. However, MALDI-MSI quantification (quantitative mass spectrometry imaging, QMSI) is an emerging technique, and needs to be further developed for practicable and convenient use in drug discovery research. In this study, we developed a reliable QMSI method for quantification of clozapine (antipsychotic drug) and dopamine and its metabolites in the rat brain using MALDI-MSI. An improved mimetic tissue model using powdered frozen tissue for QMSI was established as an alternative method, enabling the accurate quantification of clozapine levels in the rat brain. Furthermore, we used the improved method to evaluate drug-induced fluctuations in the concentrations of dopamine and its metabolites. This method can quantitatively evaluate drug localization in the brain and drug-induced changes in the concentration of endogenous metabolites, demonstrating the usefulness of QMSI. [ABSTRACT FROM AUTHOR]

A new analytical technique for detection of organic compounds using inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) is described. Volatile organic compounds (VOCs) were introduced into the collision/reaction cell (CRC), instead of through the ICP ion source, and the molecules were ionised through an ion reaction, induced by collision with the primary ions (Ar+) produced in the ICP. The ionisation characteristics of this new approach were investigated by mass spectrometric analysis of eight VOCs (i.e., benzene, toluene, ethyl acetate, methyl butyrate, ethyl butyrate, pentyl acetate, pyridine, and 2-methylfuran). These molecules were detected as molecular ions (M+), protonated ions ([M + H]+), or deprotonated ions ([M − H]+), demonstrating that soft ionisation was achieved by the present ionisation protocol using ICP-MS/MS. In addition, a volatile selenium-containing organic compound, dimethyl diselenide (Se2(CH3)2), was also analysed to investigate the feasibility of this ionisation protocol to achieve soft and hard ionisation simultaneously. Several Se-related ions such as Se+, SeH+, Se2+, [SeCH3]+, and [Se2CH3]+, together with [Se2(CH3)2]+, were observed, suggesting that while soft ionisation was possible, ion reaction-induced-fragmentation and hard ionisation also occurred. To demonstrate the analytical capability of the present technique, volatile components released from coffee beans were subjected to the present mass spectrometric analysis. Many ion peaks originating from VOCs were detected from the coffee beans. The data obtained here demonstrated that ICP-MS equipped with a CRC can become an effective tool for analyzing both elements and molecules. [ABSTRACT FROM AUTHOR]

Materials performance is primarily influenced by chemical composition, making compositional analysis (CA) essential in materials science. Traditional quantitative mass spectrometry, which deconvolutes analyte spectra into reference spectra, struggles with reactive systems where spectral variations occur, such as peak shifts and new peak emergences. Additionally, obtaining reference spectra for all pure constituents is often impractical. To address these challenges, I propose nonlinear reference-free quantitative mass spectrometry (NL-RQMS). This method simultaneously determines composition, reference spectra, and nonlinear interaction effects directly from a spectral dataset of mixtures, eliminating the need for prior reference spectra. In a benchmark test on ternary reactive polymers of epoxy and amines, NL-RQMS inferred compositions with an error margin of just 3 wt%, significantly outperforming the 6 wt% error margin of linear RQMS. The inferred interaction terms clearly indicate in situ reactions between epoxy and amine moieties. This framework enables accurate compositional analysis without prior knowledge of the constituents, even in systems with interactive components, and holds significant potential for applications such as grading recycled plastics, where pristine materials, degradation compounds, and stabilizers interact complexly, causing nonlinear spectral distortions. [ABSTRACT FROM AUTHOR]

The formation of bacterial biofilms in the human body and on medical devices is a serious human health concern. Infections related to bacterial biofilms are often chronic and difficult to treat. Detailed information on biofilm formation and composition over time is essential for a fundamental understanding of the underlying mechanisms of biofilm formation and its response to anti-biofilm therapy. However, information on the chemical composition, structural components of biofilms, and molecular interactions regarding metabolism- and communication pathways within the biofilm, such as uptake of administered drugs or inter-bacteria communication, remains elusive. Imaging these molecules and their distribution in the biofilm increases insight into biofilm development, growth, and response to environmental factors or drugs. This systematic review provides an overview of molecular imaging techniques used for bacterial biofilm imaging. The techniques included mass spectrometry-based techniques, fluorescence-labelling techniques, spectroscopic techniques, nuclear magnetic resonance spectroscopy (NMR), micro-computed tomography (µCT), and several multimodal approaches. Many molecules were imaged, such as proteins, lipids, metabolites, and quorum-sensing (QS) molecules, which are crucial in intercellular communication pathways. Advantages and disadvantages of each technique, including multimodal approaches, to study molecular processes in bacterial biofilms are discussed, and recommendations on which technique best suits specific research aims are provided. [ABSTRACT FROM AUTHOR]

Laser desorption/ionization mass spectrometry (LDI-MS) techniques have emerged as powerful tools for analyzing compounds of natural and synthetic origins. However, conventional methods like matrix-assisted laser desorption/ionization (MALDI) often suffer from background signals and limited detection capabilities, particularly for low molecular weight compounds. Surface-assisted laser desorption/ionization (SALDI) methodologies, utilizing nanomaterials, have shown promise in overcoming these limitations. Gold nanoparticles (AuNPs) are among the most widely utilized nanomaterials in SALDI-MS applications. AuNPs offer several advantages over traditional MALDI matrices, such as reduced interference, internal calibration potential, and enhanced stability. They have been successfully employed for analyzing a diverse range of analytes, from low molecular weight (LMW) compounds to peptides, proteins, and biological entities. This paper provides a comprehensive overview of various synthesis methods, morphological properties, and comparisons with conventional MALDI matrices. Furthermore, it explores the application of AuNPs for analyzing LMW compounds and imaging various surfaces using LDI-MS techniques. [ABSTRACT FROM AUTHOR]

Objectives: Biosimilars are anticipated to be widely used in the treatment of rheumatoid arthritis (RA), owing to their cost efficiency; LBEC0101 was the first etanercept (ETN) biosimilar approved in Japan. However, there are limited real-world data comparing its safety and effectiveness with those of a reference product. Methods: This study used data from the Kyoto University Rheumatoid Arthritis Management Alliance cohort, including patients with RA who received ETN therapy—ETN reference product (ETN-RP) or LBEC0101—between 2015 and 2021. Serum ETN levels were measured using liquid chromatography–tandem mass spectrometry. Results: The 1-year continuation rates of ETN-RP and LBEC0101 were 58.7% and 74.4%, respectively. Effectiveness of treatment was evaluated in 18 patients; both products significantly reduced the 28-joint RA disease activity score and erythrocyte sedimentation rate (DAS28-ESR). Moreover, to determine equivalence, we analysed 11 patients who switched from ETN-RP to LBEC0101; the DAS28-ESR and serum ETN levels before and after switching were not significantly different. Conclusions: This real-world cohort study confirmed that the biosimilar of ETN, LBEC0101, was comparable to the reference product in terms of continuation rate, effectiveness at initiation of introduction, and effect persistence before and after switching in clinical practice. [ABSTRACT FROM AUTHOR]

As one of the most common tree species, birch wood (Betula pendula Roth) is widely used as a material of furniture and decoration indoors for its many excellent characteristics. Whereas there are some studies on the volatile composition of the essential oil from the leaves of Betula pendula Roth, only limited information is available on the odor-active constituents of birch wood. To close this gap, the odorants of birch wood were investigated by means of instrumental and sensory analyses, including techniques such as gas chromatography-flame ionization detection/olfactometry, high-resolution gas chromatography-mass spectrometry, and heart-cut two-dimensional high-resolution gas chromatography-mass spectrometry/olfactometry. Overall, a total of 20 odorants was (tentatively) identified on the basis of their respective odor qualities and retention indices and mass spectra by comparison with reference compounds. It was shown that birch wood odor is dominated by a series of terpenes, terpenoids and phenyl compounds originating from the degradation of lignin and aldehydes, ketones and acids originating from fatty acid degradation. By a sensory evaluation of the birch wood, the predominant odor attributes were determined to be earthy, pencil-like, corky/mouldy, grassy, fatty, fruity, green tea-like, herb-like, vanilla-like, and vinegar-like. [ABSTRACT FROM AUTHOR]

To determine the pharmacokinetic profile of methadone after intravenous (IV) and subcutaneous (SC) administration in domestic ferrets (Mustela putorius furo). Crossover experimental study. A group of eight healthy adult ferrets weighing 1.01 ± 0.23 kg (mean ± standard deviation). Methadone hydrochloride (0.3 mg kg–1) was injected IV or SC to each ferret with a 3 week washout period. Blood samples were collected via a jugular catheter before and 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240, 360 and 480 minutes after drug administration. Liquid chromatography–tandem mass spectrometry was used to determine plasma methadone concentrations. A nonlinear mixed effects model was used to analyze the data. After IV injection, systemic clearance (Cl ss) and volume of distribution (Vd ss) were 78.9 mL min–1 kg–1 and 9.8 L kg–1, respectively. Elimination half-life was 2.0 hours and SC bioavailability was fixed at 1. The maximum observed plasma concentration after SC injection was 92.1 ± 76.8 ng mL–1. Behavioral changes were observed after both routes. The pharmacokinetic profile of IV methadone was characterized by a high Cl ss and large Vd ss , with high bioavailability and absorption rate after SC administration. Half-life was short and mean plasma methadone concentrations stayed above the minimum effective concentration (MEC) reported in humans only after SC administration for 5 minutes, but remained above that reported in dogs for 45 minutes following both routes. Further studies investigating the MEC and pharmacodynamics of methadone in ferrets are warranted. [ABSTRACT FROM AUTHOR]