High-resolution Tandem Mass Spectrometry Research Articles

Comparative Proteomics of ccRCC Cell Lines to Identify Kidney Cancer Progression Factors.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, accounting for approximately 75% of kidney cancers. The objective of this study was to identify novel progression markers for ccRCC based on proteomics, with the goal of stage determination and early diagnosis of kidney cancer patients. We performed quantitative global proteomics coupled with Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and high-resolution tandem mass spectrometry on kidney-derived cells, including HEK-293, 786-O (primary ccRCC), and Caki-1 (metastatic ccRCC) cells, to investigate the novel progression factors of ccRCC. In this study, a total of 1,106 proteins were quantified. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for differentially expressed proteins (DEPs) that were increased in ccRCC cells compared to HEK-293 cells. Ultimately, 99 DEPs including 75 up-regulated and 24 down-regulated proteins, that were significantly altered in both ccRCC cells, were identified. Among DEPs, vimentin was identified as the most significantly changed protein. Its increased expression in ccRCC was verified through immunoblotting in ccRCC cell lines and immunohistochemistry in kidney tumors. From the global proteomics data detected in ccRCC, we propose 99 DEPs including vimentin as progression factors.

Improved quality control of allergen products: Assessing the molecular allergen composition by mass spectrometry.

Natural allergen sources contain a variety of allergens, against which allergic subjects have developed individual sensitization profiles. Ideal allergen products for skin prick testing (SPT) and allergen immunotherapy (AIT) should contain the complete set of allergens of the respective allergen sources to cover all sensitization profiles. However, commercially available allergen products were shown to vary regarding their allergen composition. The qualitative allergen composition of different SPT and AIT products produced from pollen of grasses, birch, mugwort and from house dust mites was assessed by a consistent high-resolution liquid chromatography-coupled tandem mass spectrometry method (LC-MS/MS). All major, mid-tier and most minor allergens were detected in each of the investigated three batches of SPT and AIT products, demonstrating the completeness of the allergen composition and a high degree of batch-to-batch consistency. This is the first study using a single consistent high-resolution LC-MS/MS method to provide solid data on the qualitative allergen composition of SPT and AIT products manufactured from various common allergen sources. The applied method showed high reliability in qualitative batch-to-batch consistency testing and can be performed fast and with high throughput. High-resolution LC-MS/MS is applicable for process development and quality control to ensure market availability of allergen products corresponding to the composition of the respective natural allergen sources.

Discovery and Heterologous Expression of Trilenodin, an Antimicrobial Lasso Peptide with a Unique Tri-Isoleucine Motif.

Lasso peptides are an increasingly relevant class of peptide natural products with diverse biological activities, intriguing physical properties, and unique chemical structures. Most characterized lasso peptides have been from Actinobacteria and Proteobacteria, despite bioinformatic analyses suggesting that other bacterial taxa, particularly those from Firmicutes, are rich in biosynthetic gene clusters (BGCs) encoding lasso peptides. Herein, we report the bioinformatic identification of a lasso peptide BGC from Paenibacillus taiwanensis DSM18679 which we termed pats. We used a bioinformatics-guided isolation approach and high-resolution tandem mass spectrometry (HRMS/MS) to isolate and subsequently characterize a new lasso peptide produced from the pats BGC, which we named trilenodin, after the tri-isoleucine motif present in its primary sequence. This tri-isoleucine motif is unique among currently characterized lasso peptides. We confirmed the connection between the pats BGC and trilenodin production by establishing the first Bacillus subtilis 168-based heterologous expression system for expressing Firmicutes lasso peptides. We finally determined that trilenodin exhibits potent antimicrobial activity against B. subtilis and Klebsiella pneumoniae, making trilenodin the first characterized biologically active lasso peptide from Firmicutes. Collectively, we demonstrate that bacteria from Firmicutes can serve as high-potential sources of chemically and biologically diverse lasso peptides.

Integrating Epoxidation, High-Resolution Mass Spectrometry and Ultraviolet Spectroscopy to Unravel the Complex Profile of Boswellic Acids and Related Compounds in the Boswellia serrata Gum Resin Extract

The chemical characterization of natural products is often a complex task that demands powerful analytical techniques. Liquid chromatography with high-resolution tandem mass spectrometry (HRMS/MS) is often employed, yet it can face hard challenges when isomeric species are present, and reference standards are lacking. In such cases, the confidence level in compound identification can be significantly improved by the collection of orthogonal information on target analytes. In this work, 23 key compounds in Boswellia serrata extract (BSE), 12 of which correspond to boswellic acids (BAs) and 11 to triterpenoidic acid isomers, were identified by combining RPLC followed by serial UV and ESI(-)-FTMS and FTMS/MS detections with the evaluation of the reactivity towards C=C bond epoxidation with meta-chloroperoxybenzoic acid (m-CPBA), proposed as a fast chemical tool to gather information about C=C bond steric hindrance, a key structural feature of BAs and related compounds. The interpretation of UV spectra acquired after chromatographic separation corroborated the identification of the substitution patterns of enonic and dienic residues in ketoboswellic and dehydroboswellic acids. Moreover, MS/MS based on higher-energy collision-induced dissociation (HCD) unveiled new fragmentation pathways, providing important structural details on target analytes. The integrated approach developed during this study might pave the way for a deeper understanding of the BSE bioactive properties. Moreover, it can be considered an example of a more general strategy for the analysis of complex mixtures of natural compounds including also isomeric species.

Trace detection of styphnate from pre- and post-blast samples by hydrophilic interaction chromatography with tandem mass spectrometry.

Although ubiquitous in explosives and ammunition, few trace methods for detection of heavy metal-containing primary explosives from forensic samples are currently in practice. Extracts of cotton swabs or direct sampling of items were cleaned up using solid-phase extraction to remove heavy metal contaminants (i.e., lead) while retaining the organic styphnate component. The styphnate was chromatographically separated using hydrophilic interaction chromatography (HILIC) and detected via high-resolution tandem mass spectrometry (MS/MS) using a sensitive, targeted approach in five minutes or less. A mass spectrometric method for the detection of styphnate, including limit of detection (LOD), sample stability, and interferences was developed. We present a validated method for the extraction, separation, and detection of styphnate from lead(II) styphnate with an estimated LOD of 257 ppt (pg/mL). We detail an improved LOD relative to previous reports for trace detection of styphnate and, for the first time to our knowledge, the post-blast analysis of styphnate.

DEVELOPING CEREBROSPINAL flUID METABOLOMIC BIOMARKERS FOR MINIMAL RESIDUAL DISEASE AND RELAPSE IN EPENDYMOMA AND MEDULLOBLASTOMA

Abstract AIMS Relapse rates of 30% and 50% in medulloblastoma and ependymoma lead to poor outcomes for paediatric brain tumours. Occurring usually within two years, relapses imply minimal residual disease (MRD) is present below the imaging detection threshold. Patient outcomes might be improved by diagnosing MRD using a sensitive, minimally invasive cerebrospinal fluid (CSF) liquid biopsy, as CSF is proximal to tumour. Metabolites arising from aberrant cancer metabolism may be biomarkers in CSF. METHOD With only 25µl CSF, extracted with 75µl methanol, liquid chromatography coupled with tandem high-resolution mass spectrometry (LC-MS/MS) could differentiate these paediatric brain tumours (n=47; day 14 post-surgery) from controls (n=49). RESULTS Fourteen metabolites were increased in abundance in tumours and four decreased compared to controls. These included amino acids and derivatives (n=8), metabolites in one carbon metabolism (n=1), carnitine cycle (n=2), aerobic glycolysis (n=2), purine catabolism (n=2), ascorbate degradation (n=1) and creatinine synthesis (n=2). From those, betaine/creatinine ratio gave an area under the curve (AUC) of 0.96 (excellent) in receiver operating curve analysis. Using a cut-off ratio of 0.1775 for discriminating brain tumours from controls, had sensitivity and specificity of 92% and 89%. Specifically identifying tumours, a ratio of betaine/threonate with cut-off 2.1514 discriminated ependymomas from controls (sensitivity/specificity 92%/91%). A ratio of malic acid/hypoxanthine with cut-off 0.04429 discriminated medulloblastomas from controls (sensitivity/specificity 100%/98%). Medulloblastomas (n=14) were largely similar to ependymomas (n=33); nevertheless, three metabolites met the criteria of corrected p<0.05. A ratio of glutarate semialdehyde/malic acid with cut-off 0.04243 discriminated ependymomas from medulloblastomas (sensitivity/specificity 79%/82%) in a good model (AUC 0.85). CONCLUSION This is the first report of CSF metabolites in ependymoma. Ratios between metabolites levels could be useful as biomarkers to detect brain tumours and distinguish brain tumour type specifically and sensitively. Further work to develop a liquid biopsy test for the detection of MRD is ongoing.

Guidelines for Identifying the Structure of Heavy Phenolics in Lignin Depolymerization by Using High-Resolution Tandem Mass Spectrometry.

The efficient conversion of lignin contributes to reducinghuman reliance on fossil energy. As a complicated biopolymer,studies on the mechanismof lignin depolymerization is limited by inadequate structural identification of high molecular weight (MW) products like heavy phenolics. Up to now,no individual method can generate both MW and structural information in operando conditions. As a promising approach, tandem mass spectrometry (MS/MS) techniques can provide structural information via the dissociation of target ions. In this study, MS/MS technique was performed both in offline and in-situ mode during lignin depolymerization. The fundamental guidelines based on MS/MS dissociation principles for typical inter-unit linkages like β-O-4, 5-5, β-β, β-5, and β-1 were well established. Based on that, major phenolic dimers are successfully identified, including chemical formula and types of inter-unit linkages. More significantly, real-time monitoring of structural evolution was achieved by applying in-situ MS/MS analysis during lignin depolymerization. The results show the different evolution pathways of isomers with same chemical formula, confirming that structural changes during lignin depolymerization is common and obvious. Overall, this study develops an advanced strategy for the full-view analysis of lignin depolymerization, achieving the static analysis of composition and structure, both monitoring the dynamic evolution of structures.

Sub- and supratherapeutic efavirenz plasma concentrations with risk for HIV therapy failure are mainly genetically explained in Ugandan children: The prospective GENEFA cohort study.

Interindividual variations in efavirenz (EFV) plasma concentrations are extensive, but paediatric data on its consequences for viral control are scarce. The aim of this study was to explore the role of genetic variation in achieving therapeutic efavirenz plasma concentrations in a cohort of Ugandan children and the linkage between genetic CYP2B6 variants, EFV plasma variability, viral resistance and viral outcome. Ninety-nine treatment-naïve children, aged 3-12 years and living with HIV, were followed for 24 weeks after ART initiation assessing mid-dose efavirenz plasma concentrations, HIV RNA, HIV drug resistance and adherence. Polymorphisms in genes coding for drug-metabolizing enzymes were genotyped. Efavirenz concentrations were determined by liquid chromatography coupled with high-resolution tandem mass spectrometry. Metabolizer phenotype was predicted from composite genotypes of CYP2B6 (c.516G>T and c.983 T>C). A mixed effects restricted maximum likelihood regression model was used to identify important factors for efavirenz exposure. Efavirenz plasma concentrations were below the therapeutic interval (1000-4000 mg/mL) in 12-17% and above in 21-24% of measurements. Eight children had persisting subtherapeutic concentrations, five of which failed virologically and three acquired at least one new resistant mutation. Multivariate modelling explained 70% of interindividual variation in plasma concentration, with treatment duration, adherence, CYP2B6c.136A>G, and metabolizer phenotype as independent predictors of EFV concentration. In univariate analysis, metabolizer phenotype explained 50% of interindividual variation. Metabolizer phenotype explained 50% of interindividual variation in efavirenz plasma concentration. Autoinduction was not confirmed and >33% of the concentrations were outside the therapeutic interval. Subtherapeutic concentrations worsened virological resistance and outcomes. Genotype-based dosing may help avert both sub- and supratherapeutic efavirenz plasma concentrations in Ugandan children.

Sociodemographic and dietary predictors of maternal and placental mycoestrogen concentrations in a US pregnancy cohort.

Zearalenone (ZEN) is a mycotoxin contaminating grains and processed foods. ZEN alters nuclear estrogen receptor α/β signaling earning its designation as a mycoestrogen. Experimental evidence demonstrates that mycoestrogen exposure during pregnancy is associated with altered maternal sex steroid hormones, changes in placental size, and decreases in fetal weight and length. While mycoestrogens have been detected in human biospecimens worldwide, exposure assessment of ZEN in US populations, particularly during pregnancy, is lacking. To characterize urinary and placental concentrations of ZEN and its metabolites in healthy US pregnant people and examine demographic, perinatal, and dietary predictors of exposure. Urine samples were collected in each trimester from pregnant participants in the UPSIDE study and placenta samples were collected at delivery (Rochester, NY, n = 317). We used high performance liquid chromatography and high-resolution tandem mass spectrometry to measure total urinary (ng/ml) and placental mycoestrogens (ng/g). Using linear regression and linear mixed effect models, we examined associations between mycoestrogen concentrations and demographic, perinatal, and dietary factors (Healthy Eating Index [HEI], ultra-processed food [UPF] consumption). Mycoestrogens were detected in 97% of urines (median 0.323 ng/ml) and 84% of placentas (median 0.012 ng/g). Stability of urinary mycoestrogens across pregnancy was low (ICC: 0.16-0.22) and did not correlate with placental levels. In adjusted models, parity (multiparous) and pre-pregnancy BMI (higher) predicted higher urinary concentrations. Birth season (fall) corresponded with higher placental mycoestrogens. Dietary analyses indicated that higher HEI (healthier diets) predicted lower exposure (e.g., Σmycoestrogens %∆ -2.03; 95%CI -3.23, -0.81) and higher percent calories from UPF predicted higher exposure (e.g., Σmycoestrogens %∆ 1.26; 95%CI 0.29, 2.24). The mycotoxin, zearalenone (ZEN), has been linked to adverse health and reproductive impacts in animal models and livestock. Despite evidence of widespread human exposure, relatively little is known about predictors of exposure. In a pregnant population, we observed that maternal ZEN concentrations varied by maternal pre-pregnancy BMI and parity. Consumption of ultra-processed foods, added sugars, and refined grains were linked to higher ZEN concentrations while healthier diets were associated with lower levels. Our research suggests disparities in exposure that are likely due to diet. Further research is needed to understand the impacts of ZEN on maternal and offspring health.

Characterising and preventing the gut microbiota's inactivation of trifluridine, a colorectal cancer drug

The gut microbiome can metabolise hundreds of drugs, potentially affecting their bioavailability and pharmacological effect. As most gut bacteria reside in the colon, drugs that reach the colon in significant proportions may be most impacted by microbiome metabolism. In this study the anti-colorectal cancer drug trifluridine was used as a model drug for characterising metabolism by the colonic microbiota, identifying correlations between bacterial species and individuals’ rates of microbiome drug inactivation, and developing strategies to prevent drug inactivation following targeted colonic delivery. High performance liquid chromatography and ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry demonstrated trifluridine's variable and multi-route metabolism by the faecal microbiota sourced from six healthy humans. Here, four drug metabolites were linked to the microbiome for the first time. Metagenomic sequencing of the human microbiota samples revealed their composition, which facilitated prediction of individual donors’ microbial trifluridine inactivation. Notably, the abundance of Clostridium perfringens strongly correlated with the extent of trifluridine inactivation by microbiota samples after 2 hours (R2 = 0.8966). Finally, several strategies were trialled for the prevention of microbial trifluridine metabolism. It was shown that uridine, a safe and well-tolerated molecule, significantly reduced the microbiota's metabolism of trifluridine by acting as a competitive enzyme inhibitor. Further, uridine was found to provide prebiotic effects. The findings in this study greatly expand knowledge on trifluridine's interactions with the gut microbiome and provide valuable insights for investigating the microbiome metabolism of other drugs. The results demonstrate how protection strategies could enhance the colonic stability of microbiome-sensitive drugs.

An integrated strategy for in-depth profiling of N-acylethanolamines in biological samples by UHPLC-HRMS

BackgroundN-acylethanolamines (NAEs) are a class of naturally occurring bioactive lipids that play crucial roles in various physiological processes, particularly exhibiting neuroprotective and anti-inflammatory properties. However, the comprehensive profiling of endogenous NAEs in complex biological matrices is challenging due to their low abundance, structural similarity and the limited availability of commercial standards. Here, we propose an integrated strategy for comprehensive profiling of NAEs that combines chemical derivatization and a three-dimensional (3D) prediction model based on quantitative structure-retention time relationship (QSRR) using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS). ResultsAfter acetyl chloride (ACC) derivatization, the detection sensitivity of NAEs was significantly improved. We developed a QSRR prediction model to construct an in-house database for 141 NAEs, encompassing information on RT, MS1 (m/z), and MS/MS spectra. Propargylamine-labeled fatty acids were synthesized as RT calibrants across various analytical conditions to enhance the robustness of the RT prediction model. NAEs in biological samples were then in-depth profiled using parallel reaction monitoring (PRM) acquisition. This integrated strategy identified and annotated a total of 50 NAEs across serum, hippocampus and cortex tissues from a 5xFAD mouse model of Alzheimer's disease (AD). Notably, the levels of polyunsaturated NAEs, particularly NAE 20:5 and NAE 22:6, were significantly decreased in 5xFAD mice compared to WT mice, as confirmed by accurate quantitation using ACC-d0/d3 derivatization. SignificanceOur integrated strategy exhibits great potential for the in-depth profiling of NAEs in complex biological samples, facilitating the elucidation of NAE functions in diverse physiological and pathological processes.

Proteomic Profile of Circulating Extracellular Vesicles in the Brain after Δ9-Tetrahydrocannabinol Inhalation.

Given the increasing use of cannabis in the US, there is an urgent need to better understand the drug's effects on central signaling mechanisms. Extracellular vesicles (EVs) have been identified as intercellular signaling mediators that contain a variety of cargo, including proteins. Here, we examined whether the main psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), alters EV protein signaling dynamics in the brain. We first conducted in vitro studies, which found that THC activates signaling in choroid plexus epithelial cells, resulting in transcriptional upregulation of the cannabinoid 1 receptor and immediate early gene c-fos, in addition to the release of EVs containing RNA cargo. Next, male and female rats were examined for the effects of either acute or chronic exposure to aerosolized ('vaped') THC on circulating brain EVs. Cerebrospinal fluid was extracted from the brain, and EVs were isolated and processed with label-free quantitative proteomic analyses via high-resolution tandem mass spectrometry. Interestingly, circulating EV-localized proteins were differentially expressed based on acute or chronic THC exposure in a sex-specific manner. Taken together, these findings reveal that THC acts in the brain to modulate circulating EV signaling, thereby providing a novel understanding of how exogenous factors can regulate intercellular communication in the brain.

In-depth characterization of a cysteine-linked ADC disitamab vedotin by multiple LC-MS analysis methods and cutting-edge imaged capillary isoelectric focusing coupled with native mass spectrometry

The characterization of cysteine-linked antibody‒drug conjugates (ADCs) can be more challenging than that of monoclonal antibodies (mAbs) and lysine-linked ADCs because the interchain disulfide bonds are reduced for payload conjugation, and the chains are noncovalently bonded to each other. Furthermore, payload conjugation and disulfide bond reduction/scrambling may introduce additional charge heterogeneity to biomolecules. This study illustrates an innovative workflow employing multiple separation techniques and tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of disitamab vedotin, a recent-generation cysteine-linked ADC, including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX) and image capillary isoelectric focusing (icIEF). RPLC was employed for reduced chains analysis, subunit analysis and peptide mapping. IEX and icIEF were used for charge heterogeneity analysis. The innovation of the integrated methodology emphasizes the importance of cutting-edge icIEF-MS online coupling under near-native conditions to reveal the heterogeneity of disitamab vedotin.

Study of phenanthrenes from their unique mass spectrometric behavior through quantum chemical calculations to liquid chromatographic quantitation

Phenanthrenes and their derivatives have biological relevance owing to their antimicrobial, antioxidant, and cytotoxic effects on cancer cells. They can be efficiently analyzed through ultrahigh-performance liquid chromatography coupled to tandem high-resolution mass spectrometry (UHPLC–MS/HRMS). Herein, we first studied the unique fragmentation behavior of phenanthrenes based on direct infusion MS/HRMS analysis. As a newly described phenomenon, “organ pipe distribution”, we found a structural connection linking their unique fragmentation pattern to serial H radical losses. The bonds responsible for this behavior were identified through quantum chemical calculations using a stepwise approach. Furthermore, the chromatographic aspect of this study was enhanced by developing, validating, and applying a new unscheduled targeted UHPLC–MS/HRMS method for quantifying phenanthrenes in Juncus compressus herb. Targeted compounds were efficiently separated within 4 min upon utilizing the Accucore C30 column, and the unscheduled targeted analytical approach afforded five new isomers. Compounds 1 (effususol), 3 (dehydroeffusol), and 6 (7-hydroxy-1-methyl-2-methoxy-5-vinyl-9,10-dihydrophenanthrene) had their linearity limits determined within 10–5000 nM, and Compounds 2 (effusol), 4 (juncusol), 5 (effususin A), and 7 (compressin A) within 25–5000 nM. The coefficients of variation for precision ranged from 1.4 % to 15.2 %. The obtained matrix effects and accuracy values were also within acceptable ranges. Compounds 2 (effusol) and 3 (dehydroeffusol) were present in both methanolic and dichloromethanolic extracts of Plants 1 and 3 at the highest concentrations. Furthermore, the relationship between phenanthrene fingerprints, obtained through ANOVA statistical analysis of quantitative data, and the geographical location of herbs was also established.

Chemometric-based analysis and bioassay guided identification of potent compounds with intestinal motility promoting effects from Dalitong Granules

Ethnopharmacological relevanceDalitong Granules (DLT), a potent Traditional Chinese Medicine known for its ability to promote gastrointestinal motility, is widely used in clinical practice for the treatment of Functional Dyspepsia (FD). Despite the remarkable clinical efficacy of DLT, the specific components responsible for its effectiveness remains unclear. Aim of the studyThe study aimed to identify potential active ingredients of DLT for treating FD through spectrum-effect relationship analysis, multivariate statistical analysis and network pharmacology analysis. The efficacy of these identified compounds was subsequently validated using the zebrafish intestinal peristalsis model. Materials and methodsThe fingerprints of various solvent-extracted DLT were analyzed using high performance liquid chromatography coupled with tandem high-resolution mass spectrometry. The intestinal motility-promoting activities of DLT extracted by different solvents were evaluated through an intestinal propulsion test in mice. Potential therapeutic substances in DLT for treating FD were screened via chemometric analysis based on spectrum-effect relationship analysis. The correlation between the intensity of common peaks in the total ion chromatogram and the pharmacodynamic indices was assessed using multivariate statistical analysis. Additionally, given the complexity of Traditional Chinese Medicine, which comprises multiple components and targets, a network pharmacology analysis was performed to investigate the potential active ingredients in DLT. Finally, the pharmacological effects of these compounds in DLT were validated using a zebrafish intestinal motility model. ResultsThrough spectral-effect relationships analysis and network pharmacology analysis, it was determined that ten ingredients in DLT contribute to the promotion of intestinal motility. In a zebrafish intestinal motility model, it was observed that eight chemicals (excluding tetrahydropalmatine) demonstrate favorable activity of promoting gastrointestinal motility. These findings suggest that these ingredients may serve as potential therapeutic agents for improving gastric motility disorders. ConclusionsThis study employed spectral-effect relationship and network pharmacology analysis to identify the active ingredients in DLT. The findings were then validated using a zebrafish intestinal peristalsis model. These results provide a scientific foundation for the clinical application of DLT as a key traditional herbal formula for managing FD.

The blistering warfare agent O-mustard (agent T) generates protein-adducts with human serum albumin useful for biomedical verification of exposure and forms intramolecular cross-links.

The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys34 and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HRMS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide Cys34*Pro and the single amino acid residue His* were produced. The use of proteinaseK yielded Cys34*ProPhe and theuse of pepsin generated ValThrGlu48*Phe, AlaGlu230*ValSerLysLeu, and LeuGlyMet329*Phe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu230 and Met239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys34(-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu86, which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time.

Consolidating two-dimensional liquid chromatography–high-resolution tandem mass spectrometry (LC×LC-HRMS/MS) technique for the non-targeted analysis of poly- and perfluorinated substances: A trial on aqueous film-forming foams

To date, poly- and perfluoroalkyl substances (PFAS) represent a real threat for their environmental persistence, wide physicochemical variability, and their potential toxicity. Thus far a large portion of these chemicals remain structurally unknown. These chemicals, therefore, require the implementation of complex non-targeted analysis workflows using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for their comprehensive detection and monitoring. This approach, even though comprehensive, does not always provide the much-needed analytical resolution for the analysis of complex PFAS mixtures such as fire-fighting aqueous film-forming foams (AFFFs). This study consolidates the advantages of the LC×LC technique hyphenated with high-resolution tandem mass spectrometry (HRMS/MS) for the identification of PFAS in AFFF mixtures. A total of 57 PFAS homolog series (HS) were identified in 3M and Orchidee AFFF mixtures thanks to the (i) high chromatographic peak capacity (n’2D,c ~ 300) and the (i) increased mass domain resolution provided by the “remainder of Kendrick Mass” (RKM) analysis on the HRMS data. Then, we attempted to annotate the PFAS of each HS by exploiting the available reference standards and the FluoroMatch workflow in combination with the RKM defect by different fluorine repeating units, such as CF2, CF2O, and C2F4O. This approach resulted in 12 identified PFAS HS, including compounds belonging to the HS of perfluoroalkyl carboxylic acids (PFACAs), perfluoroalkyl sulfonic acids (PFASAs), (N-pentafluoro(5)sulfide)-perfluoroalkane sulfonates (SF5-PFASAs), N-sulfopropyldimethylammoniopropyl perfluoroalkane sulfonamides (N-SPAmP-FASA), and N-carboxymethyldimethylammoniopropyl perfluoroalkane sulfonamide (N-CMAmP-FASA). The annotated categories of perfluoroalkyl aldehydes and chlorinated PFASAs represent the first record of PFAS HS in the investigated AFFF samples.

Systematic Characterization of Sesquiterpenes from Dendrobium nobile through Offline Two-Dimensional Chromatography Tandem Mass Spectrometry and Target Isolation.

Dendrobium nobile is a species of the genus Dendrobium that can be used as both a medicinal herb and healthy food. The sesquiterpenes in D. nobile have attracted extensive attention in recent years. In this study, Amide × RP offline two-dimensional chromatography separation tandem high-resolution mass spectrometry combined with GNPS (Global Natural Product Social Molecular Networking) was developed for the characterization of sesquiterpenes in D. nobile. After first-dimensional amide separation, the 70% ethanol extract of D. nobile was divided into 40 fractions, which were analyzed by second-dimensional reverse-phase system separation and LTQ-Orbitrap detection. The raw data was imported into the GNPS, resulting in the efficient clustering of similar substances. Finally, 594 sesquiterpene compounds were characterized, and 25 compounds were isolated based on molecular network analysis, including six new compounds. In vitro bioassays, the isolated compounds decreased NO production in the LPS-induced microglial BV-2 cell model and the content of MDA in PC12 cells, demonstrating neuroprotective activity. These findings unraveled the underlying material and provided valuable insights into the quality control of D. nobile.

Feature-Based Molecular Network for New Psychoactive Substance Identification: The Case of Synthetic Cannabinoids in a Seized e-Liquid and Biological Samples.

The comprehensive detection of new psychoactive substances, including synthetic cannabinoids along with their associated metabolites in biological samples, remains an analytical challenge. To detect these chemicals, untargeted approaches using appropriate bioinformatic tools such as molecular networks are useful, albeit it necessitates as a prerequisite the identification of a node of interest within the cluster. To illustrate it, we reported in this study the identification of synthetic cannabinoids and some of their metabolites in seized e-liquid, urine, and hair collected from an 18-year-old poisoned patient hospitalized for neuropsychiatric disorders. A comprehensive analysis of the seized e-liquid was performed using gas chromatography coupled with electron ionization mass spectrometry, 1H NMR, and liquid chromatography coupled with high resolution tandem mass spectrometry combined with data processing based on molecular network strategy. It allowed researchers to detect in the e-liquid known synthetic cannabinoids including MDMB-4en-PINACA, EDMB-4en-PINACA, MMB-4en-PINACA, and MDMB-5F-PICA. Compounds corresponding to transesterification of MDMB-4en-PINACA with pentenol, glycerol, and propylene glycol were also identified. Regarding the urine sample of the patient, metabolites of MDMB-4en-PINACA were detected, including MDMB-4en-PINACA butanoic acid, dihydroxylated MDMB-4en-PINACA butanoic acid, and glucurono-conjugated MDMB-4en-PINACA butanoic acid. Hair analysis of the patient allowed the detection of MDMB-4en-PINACA and MDMB-5F-PICA in the two investigated hair segments. This untargeted analysis of seized materials and biological samples demonstrates the utility of the molecular network strategy in identifying closely related compounds and metabolites of synthetic cannabinoids. It also emphasizes the need for developing strategies to anchor molecular networks, especially for new psychoactive substances.

Screening of Antagonistic Bacteria against Three Aquatic Pathogens and Characterization of Lipopeptides in Bacillus cereus BA09.

Screening for antagonistic bacteria on aquatic pathogens and identification of antagonistic ingredients are essential to reduce the use of chemicals in aquaculture. In this study, strain BA09, subsequently identified as Bacillus cereus, simultaneously displayed strong antagonistic effects on Edwardsiella tarda, Vibrio harveyi, and Streptococcus anisopliae in the initial screening and rescreening. In addition, the methanol extract of BA09 was subjected to antibacterial activity verification and one-dimensional (1D) reversed-phase liquid chromatography (RPLC) preparation. A total of 27 fractions were collected, 6 of which were subjected to two-dimensional (2D) RPLC separation and tracked as antibacterial. A total of 14 lipopeptides that included 9 fengycin homologs, 3 bacillomycin homologs, and 2 surfactin homologs were identified by tandem high-resolution mass spectrometry. Through characterization of the antibacterial substance in Bacillus cereus BA09, which simultaneously inhibited E. tarda, V. harveyi, and S. agalactiae, the current study provides a theoretical basis for the development of antibacterial drugs in aquaculture.