Mass Spectrometric Analysis Research Articles

Spectroscopic properties of a fluorescent pyrene-quinoline-based bisoxime-type sensor and its application to the detection of Cu2+ ion in real water samples

Fluorescent sensors are efficient and convenient tools for metal ions analysis. Herein, a new fluorescent pyrene-quinoline-based bisoxime-type sensor PQS, possessing the monomer to excimer emission switching feature, was synthesized and the sensing ability toward Cu2+ was throughly studied. Interestingly, the blue emission of monomer PQS in dilute solution converted to the green fluorescence of excimer in aggregated state. Meanwhile, the blue fluorescence emission of sensor PQS quenched in the existence of Cu2+ with no apparent response toward other competitive cations. The plausible complexation ratio 1:1 of sensor PQS to Cu2+ was substantiated by Job's plot, mass spectrometric analysis combined with the density functional theory calculation. Furthermore, PQS coated paper strips were prepared and used for the rapid and convenient monitoring Cu2+ in a visible manner.

Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells.

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

A computational and experimental study of cis-trans isomeric pesticides based on collision-induced dissociation of high-resolution mass spectrometry.

Pesticide isomers are widely available in agricultural production and may vary widely in biological activity, potency, and toxicity. Chromatographic and mass spectrometric analysis of pesticide isomers is challenging due to structural similarities. Based on liquid chromatography time-of-flight mass spectrometry, identification of cis-trans isomeric pesticides was achieved through retention time, characteristic fragment ions, and relative abundance ratio. Furthermore, theoretical and basic research has been conducted on the differences in characteristic fragment ions and their relative abundance ratios of cis-trans isomers. On the one hand, the cleavage pathways of six cis-trans isomers were elucidated through collision-induced dissociation to explain different fragment ions of the isomers. On the other hand, for those with the same fragment ions but different abundance ratios, energy-resolved mass spectrometry combined with computational chemical density functional theory in terms of kinetics, thermodynamics, and bond lengths was employed to explain the reasons for the differences in characteristic fragment ions and their abundance ratios. A high-resolution mass spectrometry method was developed for the separation and analysis of cis-trans isomers of pesticides in traditional Chinese medicine Radix Codonopsis, and six pesticide isomers were distinguished by retention time, product ions, and relative abundance ratios. The limits of quantification of the six pesticides were up to 10μg/kg, and the linear ranges of them were 10-200 μg/kg, with coefficients of determination (R2) > 0.99, which demonstrated the good linearity of the six pesticides. The recoveries of the pesticides at spiked concentrations of 10, 20, and 100 μg/kg reached 70-120% with relative standard deviations ≤20%. It was demonstrated that the application of the method was well suited for accurate qualitative and quantitative analysis for isomers with different structures, which could avoid false-negative results caused by ignoring other isomers effectively.

Long term online desalting analysis of MS/LC-MS using thermal assisted recrystallization ionization

Mass spectrometric analysis of non-volatile salts containing samples remains challenging due to salt-induced ion suppression and contamination. This challenge is even more pronounced for a liquid chromatography-mass spectrometry analysis, where the accumulation of salts in the transmission system poses an ongoing problem. In this study, a novel thermal assisted recrystallization ionization mass spectrometry (TARI-MS) device was developed to achieve efficient on-line desalting and prolonged analysis of saline samples. The core component of this device was a heated plate positioned between the electrospray unit and the MS inlet. The desalting mechanism was demonstrated as the spontaneous separation of target molecules from salts during the “crystallization” process. After optimization, the angle between the nebulizer and the heated plate was 45°; the distance between the front end of the heated plate and the MS inlet was 2 mm; the distance between the front edge of the heated plate and the center of the sample spray projected onto the heating plate was 3 mm; the distance between the emitter of nebulizer and the heated plate was 3 mm. TARI-MS realized direct analysis of eight drugs dissolved in eight commonly used non-volatile salts solutions (up to 0.5 mol/L). The high sensitivity, repeatability, linearity, accuracy, and intra- and inter-day precision of TARI-MS confirm its reliability as a robust tool for the analysis of saline samples. Furthermore, TARI-MS allowed continuous analysis of salty eluates of LC for up to nearly 1 h without maintenance and verified the feasibility of LC-MS analysis through detecting a five-drug mixture and a crude aripiprazole product. Finally, six impurities in the crude aripiprazole product were successfully detected by LC-TARI-MS. The established method holds promise for applications across academic and pharmaceutical domains.

Novel serum protein biomarker panel for early diagnosis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Late presentation of disease at the time of diagnosis is one of the major reasons for dismal prognostic outcomes for PDAC patients. Currently, there is a lack of clinical biomarkers, which can be used to diagnose PDAC patients at an early resectable stage. This study performed proteomic mass spectrometry to identify novel blood-based biomarkers for early diagnosis of PDAC. Serum specimens from 88 PDAC patients and 88 healthy controls (60 discovery cohort and 28 validation cohort) were analyzed using data independent acquisition high resolution mass spectrometry to identify candidate biomarker proteins. A total of 249 proteins were identified and quantified by the mass spectrometric analysis. Six proteins were markedly (>1.5 fold) and significantly (p < .05; q < 0.1) increased in PDAC patients compared to healthy controls in discovery cohort. Notably, four of these six proteins were significantly upregulated in an independent validation cohort. The top three upregulated proteins (i.e., Polymeric Immunoglobulin Receptor [PIGR], von Willebrand Factor [vWF], and Fibrinogen) were validated using enzyme linked immunosorbent assay, which led to selection of PIGR and vWF as a diagnostic biomarker panel for PDAC. The panel showed high ability to diagnose early stage (stage I and II) PDAC patients (area under the curve [AUC]: 0.8926), which was further improved after the addition of clinically used prognostic biomarker (Ca 19-9) to the panel (AUC: 0.9798). In conclusion, a novel serum protein biomarker panel for early diagnosis of PDAC was identified.

Abstract 937: Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe

Abstract Translation of precision oncology data into feasible precision therapies for hard-to-cure childhood, adolescent and young adult malignancies remains a significant challenge. Identifying therapeutic targets at the protein and pathway level and demonstrating treatment response in personalized models hold great promise, particularly for combination therapies, but may be considered complex and time consuming. Here, we present the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how proteomics combined with rapid patient-derived models can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. Elevated SHMT2 levels were validated by immunohistochemistry and compared to levels across AYA tumors. Based on the suitability for an innovative therapy trial, we prioritized single-agent sertraline, a commercially available anti-depressant medication that inhibits SHMT2, and confirmed a positive drug response in both chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Retrospective expansion in a murine xenograft enabled metabolic tracing on isolated SETTLE- patient-derived xenograft cells using 13C6-glucose confirming SHMT2 activity and response to in vitro treatment. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial of sertraline was initiated by the patient but stopped after 8 weeks after evidence of progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Combining sertraline with the common antibiotic trimethoprim, resulted in enhanced growth inhibition of SETTLE cells in the larval zebrafish xenografts. Significance: Overall, we demonstrate that proteomics and personalized xenograft models may provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact clinical practice. Citation Format: Georgina D. Barnabas, Tariq A. Bhat, Verena Goebeler, Pascal Leclair, Nadine Azzam, Nicole Melong, Jason N. Berman, Jennifer A. Chan, Donna L. Senger, Seth Parker, Christopher A. Maxwell, Gregor S. Reid, Jonathan Bush, Caron Strahlendorf, Rebecca Deyell, C James Lim, Philipp F. Lange. Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 937.

Exposure of Male Farmers and Nonfarmers to Neonicotinoid Pesticides in the South-West and Littoral Regions of Cameroon: A Comparative Study.

Pesticides, especially the newly developed neonicotinoids, are increasingly used in many countries around the world, including Cameroon, to control pests involved in crop destruction or disease transmission. Unfortunately, the pesticides also pose tremendous environmental problems because a predominant amount of their residues enter environmental matrices to affect other nontargeted species including humans. This therefore calls for continuous biomonitoring of these insecticides in human populations. The present study sought to assess the neonicotinoid insecticide exposures in two agrarian regions of Cameroon, the South-West region and Littoral region. The study involved 188 men, including 125 farmers and 63 nonfarmers. Spot urine samples were obtained from these subjects and subjected to liquid chromatographic-tandem mass spectrometric analysis for concentrations of neonicotinoid compounds, including acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram, thiamethoxam, and N-dm-acetamiprid. Neonicotinoid compounds were detected in all study participants, and residues of all the screened pesticides were detected among participants. N-dm-Acetamiprid and imidacloprid were the most prevalent among the subjects (100.0% and 93.1%, respectively), whereas nitenpyram was less common (3.2%). The median values of imidacloprid and total urinary neonicotinoid concentrations were elevated among farmers (0.258 vs. 0.126 µg/L and 0.829 vs. 0.312 µg/L, respectively). Altogether the findings showed that both the farmer and nonfarmer study populations of Cameroon were exposed to multiple residues of neonicotinoids, with relatively higher levels of pesticides generally recorded among farmers. Although exposure levels of the neonicotinoids were generally lower than their respective reference doses, these results warrant further research on the health risk evaluation of multiple residues of the pesticides and reinforcement of control measures to minimize the exposure risks, especially among farmers. Environ Toxicol Chem 2024;43:952-964. © 2024 SETAC.

A fully validated LC‑MS/MS method for quantifyingbevacizumab in plasma samples from patients with NSCLC and its implications in therapeutic drug monitoring.

Given the increasing use of bevacizumab in combinatorial drug therapy for a multitude of different cancer types, there is a need for therapeutic drug monitoring to analyze the possible correlation between drug trough concentration, and therapeutic effect and adverse reactions. An ultra-performance liquid chromatography tandem-mass spectrometry method was then developed and validated to determine bevacizumab levels in human plasma samples. Chromatographic separation was achieved on a Shimadzu InertSustainBio C18 HP column, whereas subsequent mass spectrometric analysis was performed using a Shimadzu 8050CL triple quadrupole mass spectrometer equipped with an electro-spray ionization source in the positive ion mode. In total, three multiple reaction monitoring transitions of each of the surrogate peptides were chosen with 'FTFSLDTSK' applied as the quantification peptide whereas 'VLIYFTSSLHSGVPSR' and 'STAYLQMNSLR' were designated as the verification peptides using the Skyline software. This analytical method was then fully validated, with specificity, linearity, lower limit of quantitation, accuracy, precision, stability, matrix effect and recovery calculated. The linearity of this method was developed to be within the concentration range 5-400 µg/ml for bevacizumab in human plasma. Subsequently, eight patients with non-small cell lung cancer (NSCLC) were recruited and injected with bevacizumab over three periods of treatment to analyze their steady-state trough concentration and differences. To conclude, the results of the present study suggest that bevacizumab can be monitored in a therapeutic setting in patients with NSCLC.

Abstract 3940: Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8

Abstract Breast cancer is a highly complex multifactorial disease, which can be driven by the aberrant regulation of different signaling pathways including receptor tyrosine kinase (RTK) signaling. Notably, while RTKs have been classically described as transmembrane receptors, they can also translocate to the nucleus, even though the functional importance of this process remains largely unexplored for most RTKs. Here, we report a novel role for the nuclear form of the RTK human epidermal growth factor receptor 3 (HER3) in driving primary breast cancer growth and metastasis. Firstly, using different patient-derived organoids (PDOs) established from metastatic breast cancer patients, we demonstrated the robust translocation of the activated phosphorylated HER3 receptor (pHER3) from the plasma membrane to the nucleus in response to its ligand Neuregulin 1 (NRG1). Interestingly, the nuclear translocation was observed not only in the HER2-positive breast cancer subtype but also in luminal and triple-negative breast cancer-derived cells. In order to decipher the functional role of nuclear HER3 (nHER3), we identified and mutated its nuclear localization signal, resulting in decreased nuclear translocation of the receptor while its membrane form remained intact. This reduction in nHER3 remarkably impeded primary tumor growth and metastatic spread in different patient-derived xenograft (PDX) models. Conversely, selective overexpression of HER3 in the nucleus led to increased primary tumor growth and metastatic burden in vivo. In order to decipher the mechanism of action of nHER3, we performed co-immunoprecipitation followed by global mass spectrometric analysis to identify the protein binding partners of the receptor in the nucleus. Specifically, nHER3 was found to interact with different transcription factors such as the AP-2 family of transcription factors and with major chromatin remodeling complexes like the nucleosome remodeling and deacetylase (NuRD) complex, implicating its role in transcriptional regulation. Furthermore, gene expression analysis of PDOs expressing the wild-type or the mutated form of HER3 revealed the modulation of several key growth regulators such as early growth response 3 (EGR3) by nHER3. The nHER3-EGR3 axis further controlled downstream effectors like the immune chemoattractants CXCL1 and CXCL8. Accordingly, PDX from breast cancer cells overexpressing nHER3 showed significantly increased immune infiltration, suggesting a possible role of nHER3 in regulating the tumor microenvironment. Altogether, we report here a novel non-canonical role of the nuclear form of HER3 receptor in driving breast tumorigenesis, with key implications for our understanding and targeting of RTK signaling in breast cancer. Citation Format: Tasneem Cheytan, Roberto Würth, Nina Hahnen, Elisa Donato, Corinna Klein, Rebecca Weber, Daniele Colombo, Jeroen Krijgsveld, Martin Sprick, Andreas Trumpp. Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3940.

Abstract 2871: Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer

Abstract Purpose Statement: Alterations in the PI3K/AKT signaling pathway are common in cervical cancer. The most commonly observed mutations are in PIK3CA (26%) and PTEN (8%). However, the biological and metabolic implications of acquiring these mutations in the cervical cancer genome are poorly characterized. Materials/Methods: Isogenic cell lines with PIK3CA E545K and PTEN null mutations were generated using Crispr/CAS9. Cell viability and clonogenic cell survival assays were performed in the presence and absence of inhibitors of glutamine and asparagine metabolism. Glucose and glutamine metabolism was evaluated by stable isotope labeling with U13C glucose and13C5 glutamine in presence of glutamine antagonist DON. RNA-seq was employed to evaluate transcriptomic alterations. Results: Introduction of the PIK3CA E545K mutation and PTEN mutation in cervical tumor cells leads to an increase in AKT signaling compared to the parental (wild-type) cells. Notably, RNASeq and mass spectrometric analysis of both the tumor cells and their culture media revealed heightened dependencies on glucose and glutamine in the tumor cells carrying the PIK3CA E545K and PTEN mutations as compared to the wild-type cells. Treatment with glutamine antagonists (DON, JHU-083) and GLS inhibitors (CB-839 and IACS-6274), resulted in reduced cell viability and clonogenic survival in tumor cells with PI3K/PTEN mutation compared to wild-type. Additionally, we observed a metabolic reliance on the amino acid asparagine in tumors with PI3K/PTEN alterations when treated with DON. Asparaginase in combination with DON effectively inhibited the proliferation of human and mouse cell lines derived from primary tumors with PTEN mutations. Furthermore, the incorporation of a glutamine antagonist and asparaginase into conventional chemoirradiation further enhanced the therapeutic efficacy in cervical tumors with PI3K/PTEN alterations which are otherwise treatment resistant. Conclusions: Orthogonal evaluations using glutamine agonists, GLS inhibitors, and mass spectrometric point to a significant role played by PI3K/PTEN mutations in altering cervix tumor cell metabolism towards a more glutamine dependent phenotype. This study suggests a potential strategy for improving the treatment of PI3K-AKT-altered cervical tumors by targeting glutamine and asparagine metabolism in combination with standard therapies. These treatment strategies are currently being evaluated in vivo using a patient derived xenograft model with PIK3CA E545K mutation as well as an immunocompetent cervical tumor model in mice with PTEN deletion. Citation Format: Nishanth Noel Gabriel, Naoshad Muhammad, Kevin Cho, Kay Jayachandran, Jin Zhang, Gary Patti, Julie Schwarz. Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2871.

High-purity rayon-based carbon fiber felt prepared by halogen gas purification for superior thermal insulation and oxidation resistance

High-purity rayon-based carbon fiber (CFRay) felt holds significant promise for applications in thermal protection systems and semiconductor crystal growth furnaces. This study employs halogen gas purification and high-temperature purification techniques in the fabrication of high-purity CFRay felt. Additionally, the influence of the purification process on the thermal insulation and oxidation resistance of CFRay felt is investigated. The glow discharge mass spectrometer analysis reveals that purification at a lower temperature of 2300 °C achieves a purity level of 6.9 ppm, surpassing both purified CFRay felt without halogen at 2300 °C (56.1 ppm) and high-temperature treated CFRay felt at 3000 °C (10.4 ppm). Thermal insulation and dynamic-static oxidation resistance tests show that the properties of CFRay felt are concurrently affected by the heat treatment temperature. Compared to high-temperature purification, halogen gas purification operates at lower temperatures, preventing the graphitization of CFRay, thereby ensuring high purity while enhancing thermal insulation and demonstrating relatively superior oxidation resistance. This study offers novel insights into the preparation and performance adjustment of high-purity CFRay felt.

Diagonal Chromatography for the Isolation of O-Linked β-N-acetyl-D-Glucosaminylated (O-GlcNAcylated) Peptides in HeLa Cell Extracts

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC–MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC–MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.

Identification of New Ketamine Metabolites and Their Detailed Distribution in the Mammalian Brain.

Ketamine is a common anesthetic used in human and veterinary medicine. This drug has recently received increased medical and scientific attention due to its indications for neurological diseases. Despite being applied for decades, ketamine's entire metabolism and pharmacological profile have not been elucidated yet. Therefore, insights into the metabolism and brain distribution are important toward identification of neurological effects. Herein, we have investigated ketamine and its metabolites in the pig brain, cerebrospinal fluid, and plasma using mass spectrometric and metabolomics analysis. We discovered previously unknown metabolites and validated their chemical structures. Our comprehensive analysis of the brain distribution of ketamine and 30 metabolites describes significant regional differences detected mainly for phase II metabolites. Elevated levels of these metabolites were identified in brain regions linked to clearance through the cerebrospinal fluid. This study provides the foundation for multidisciplinary studies of ketamine metabolism and the elucidation of neurological effects by ketamine.

Isolation and Antitumoral Effect of a New Siphonochilone Derivative from African Ginger.

A new eusdesmane sesquiterpenoid, characterized as 3,5,8a-trimethyl-8-oxo-4,4a,5,6,7,8,8a,9-octahydronaphtho[2,3-b]furan-5-yl acetate (1), has been isolated from the rhizomes of the South African variety of wild ginger (Siphonochilus aethiopicus (Schweinf) B. L. Burtt). The compound was obtained by silica gel column chromatography. Its structure was elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass-spectrometric (MS) analyses, including 1D-, 2D-NMR, and HR-LCMS. We also investigated the cytotoxic effect of 1 on a panel of cancer cell lines, human breast, pancreas, lung, colon, and central nervous system cancer lines. The data are not encouraging since its antitumor effect is poor. Nonetheless, the discovery of new molecules may provide a source of new compounds with important biological effects applicable to the field of medicine.

Brominated Dioxins in Egg, Broiler, and Feed Additives: Significance of Bioassay-Directed Screening for Identification of Emerging Risks in Food.

Food authorities aim to safeguard our food. This requires sensitive analyses to guarantee detection of both banned and regulated substances at low concentrations. At the same time, broad screening methods are needed to identify new emerging risks. For this purpose, effect-based bioassays combined with mass spectrometric analyses offer an advantage. During the regular monitoring of dioxins in agricultural products, a discrepancy was observed between the results of the DR CALUX (Dioxin-Responsive Chemical Activated Luciferase gene Expression) bioassay and the confirmatory gas chromatographic high resolution mass spectrometric (GC-HRMS) analysis in egg and broiler fat samples. The response in the bioassay was high, suggesting a clear exceedance of the maximum limits of dioxins in these samples, yet regulated dioxins or dl-PCBs were not detected by GC/HRMS analysis. Ultimately, a broad screening analysis using GC-HRMS resulted in the identification of 2,3,7,8-tetrabromo-dibenzofuran (2,3,7,8-TBDF) in both egg and broiler fat. To investigate the potential source of this brominated furan contaminant, different samples were analyzed: bedding material, poultry feed, feed additives (choline chloride and l-lysine), and seaweed. The poultry feed and feed additives all contained 2,3,7,8-TBDF. Using a feed-to-food transfer model, it became clear that the poultry feed was probably the source of 2,3,7,8-TBDF in broilers and eggs through a feed additive like L-lysine or choline chloride. This study underlines the importance of using a combination of effect-based screening assays with sensitive analytical methods to detect potential new and emerging risks.

Qualitative and quantitative analysis of pesticide isomers based on collision-induced dissociation of high-resolution mass spectrometry

Pesticide isomers are widely present in agricultural production, while their biological activity, efficacy, and toxicity may vary significantly. Due to structural similarity, chromatographic and mass spectrometric analysis of isomeric pesticides are challenging. In this experiment, representative isomeric pesticides with great differences in toxicity and maximum residue limits among countries were studied. Based on collision-induced dissociation (CID) of high-resolution mass spectrometry, the fragmentation pathways of substituent position isomer, functional group position isomer, carbon frame isomer, functional group isomer, and cis–trans isomer were analyzed to attribute fragment ions. In addition, for the isomers with the same product ions and different abundance ratios, the abundance differences at different energy-resolved mass spectrometry were analyzed, which was combined with the computational chemical density functional theory to simulate the specific process of fragmentation and to explain the reasons for the differences in fragment ions and abundance ratios. Based on the above study, a high-resolution mass spectrometry method was established for the separation and analysis of pesticide isomers. Twelve pesticide isomers were distinguished by retention time, fragment ions, and relative abundance ratio. This method was applied to the detection of real fruit and vegetable samples.

Current data processing methods and reporting standards for untargeted analysis of volatile organic compounds using direct mass spectrometry: a systematic review

IntroductionUntargeted direct mass spectrometric analysis of volatile organic compounds has many potential applications across fields such as healthcare and food safety. However, robust data processing protocols must be employed to ensure that research is replicable and practical applications can be realised. User-friendly data processing and statistical tools are becoming increasingly available; however, the use of these tools have neither been analysed, nor are they necessarily suited for every data type.ObjectivesThis review aims to analyse data processing and analytic workflows currently in use and examine whether methodological reporting is sufficient to enable replication.MethodsStudies identified from Web of Science and Scopus databases were systematically examined against the inclusion criteria. The experimental, data processing, and data analysis workflows were reviewed for the relevant studies.ResultsFrom 459 studies identified from the databases, a total of 110 met the inclusion criteria. Very few papers provided enough detail to allow all aspects of the methodology to be replicated accurately, with only three meeting previous guidelines for reporting experimental methods. A wide range of data processing methods were used, with only eight papers (7.3%) employing a largely similar workflow where direct comparability was achievable.ConclusionsStandardised workflows and reporting systems need to be developed to ensure research in this area is replicable, comparable, and held to a high standard. Thus, allowing the wide-ranging potential applications to be realised.

Dependence on relative humidity in the formation of reactive oxygen species in water droplets

Water microdroplets (7 to 11 µm average diameter, depending on flow rate) are sprayed in a closed chamber at ambient temperature, whose relative humidity (RH) is controlled. The resulting concentration of ROS (reactive oxygen species) formed in the microdroplets, measured by the amount of hydrogen peroxide (H2O2), is determined by nuclear magnetic resonance (NMR) and by spectrofluorimetric assays after the droplets are collected. The results are found to agree closely with one another. In addition, hydrated hydroxyl radical cations (•OH-H3O+) are recorded from the droplets using mass spectrometry and superoxide radical anions (•O2-) and hydroxyl radicals (•OH) by electron paramagnetic resonance spectroscopy. As the RH varies from 15 to 95%, the concentration of H2O2 shows a marked rise by a factor of about 3.5 in going from 15 to 50%, then levels off. By replacing the H2O of the sprayed water with deuterium oxide (D2O) but keeping the gas surrounding droplets with H2O, mass spectrometric analysis of the hydrated hydroxyl radical cations demonstrates that the water in the air plays a dominant role in producing H2O2 and other ROS, which accounts for the variation with RH. As RH increases, the droplet evaporation rate decreases. These two facts help us understand why viruses in droplets both survive better at low RH values, as found in indoor air in the wintertime, and are disinfected more effectively at higher RH values, as found in indoor air in the summertime, thus explaining the recognized seasonality of airborne viral infections.

Phosphorus-containing stereocontrolled polyhydroxyalkanoates by yttrium-mediated ring-opening copolymerization of β-lactones

Poly(3-hydroxybutyrate) (PHB) polymers functionalized with pendent diphenyl-phosphinate moieties were prepared by the simultaneous ring-opening copolymerization (ROCOP) of racemic or enantiopure β-butyrolactone (rac- or (S)-BPLMe, respectively) with racemic-(4-oxooxetan-2-yl)methyl diphenylphosphinate (rac-BPLOP). The reactions were mediated by yttrium catalysts which tunable ancillary structure enabled preparing copolymers containing syndio-enriched (Pr up to 0.84; Pr is the probability of racemic linkage), atactic (Pr ∼ 0.50) and highly isotactic (Pr < 0.06) poly(BPLMe) (P(BPLMe), aka PHB for polyhydroxybutyrate) segments, starting from rac-BPLMe or (S)-BPLMe, respectively, as assessed by detailed NMR analyses of the copolymer samples. The amount of BPLOP incorporated within the P[(BPLMe)x-co-(BPLOP)y] samples were in line with the initial comonomer loadings (1.8–8.1 mol% vs. 2–10 mol%). The ROCOP was rather well-controlled affording linear α-isopropoxy, ω-crotonyl telechelic and cyclic random copolymers with Mn,NMR up to 15,000 g.mol−1 and ÐM = 1.34–1.95. Detailed MALDI and high-resolution ESI mass spectrometric analyses evidenced the formation of three major series of macromolecules, namely the cyclic one and two linear ones end-capped with an α-isoproxy and either an ω-hydroxy or a ω-crotonate groups; for each of these series, the populations containing y = 0, 1 or 2 BPLOP repeating units were unambiguously distinguished. The thermal characteristics of the P[(BPLMe)x-co-(BPLOP)y] copolymers were investigated by DSC and TGA, revealing in comparison to those of the parent PHB homopolymers, tunable features as a function of their molar mass, tacticity and BPLOP content.

Biochemical characterization of cardiac α-actin mutations A21V and D26N implicated in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α-actin are the cause in 1%-5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM-linked cardiac α-actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α-actin variants and wild-type protein revealed improper N-terminal processing in the Spodoptera frugiperda (Sf-9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N-terminal deletion mutants lacking the N-terminal cysteine (ΔC2). The ΔC2 wild-type construct behaved similar to porcine cardiac α-actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α-actin. In vitro motility assays and transient-kinetic studies probing the interaction of the actin variants with cardiac β-myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac β-myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild-type and p.D26N to the same level of 1100 nm s-1 .