Search

Identification of molecules in complex natural matrices relies on matching the fragmentation spectra of ions under investigation and the spectra acquired for the corresponding analytical standards. Currently, there are many databases of experimentally measured tandem mass spectrometry spectra (such as NIST, MzCloud, and Metlin), and considerable progress has been made in the development of software for predicting tandem mass spectrometry fragments in silico using combinatorial, machine learning, and quantum chemistry approaches (such as MetFrag, CFM‐ID, and QCxMS). However, the electrospray ionization molecules can be ionized at different sites (protonated or deprotonated), and the fragmentation spectra of such ions are different. Here, we are using the combination of the in‐ESI source hydrogen/deuterium exchange reaction and MSn fragmentation for the investigation of the fragmentation pathways for different protomers of organic molecules. It is shown that the distribution of the deuterium in the fragment ions reflects the presence of different protomers. For several molecules, the distribution of deuterium was traced up to the MS5 level of fragmentation revealing many unusual and unexpected effects. For example, we investigated the loss of HF from the ciprofloxacin and norfloxacin ions and observed that for ions protonated at –COOH group, the eliminating hydrogen always comes from –NH group. When ions are protonated at another site, the elimination of hydrogen with a probability of 30% occurs from the –NH group, and with a probability of 70%, it originates from other sites on the molecule. Such effects were not described previously. Quantum chemical simulation was used for the verification of the protonated structures and simulation of the corresponding fragmentation spectra. [ABSTRACT FROM AUTHOR]

Chloroformates are prevalent in the atmosphere due to their utilization as fuel additives and industrial solvents. These species may undergo interactions with atmospheric water resulting in protonated chloroformates. This study delves into the gas‐phase dissociation of these protonated species. Tandem mass spectrometry was employed to scrutinize the unimolecular dissociation of protonated methyl (1), ethyl (2), neopentyl (3), and phenyl chloroformate (4). Notably, 1 and 4 exhibited HCl loss, yielding CH3OCO+ and C6H5OCO+, respectively, with 1 additionally generating neutral methanol and ClCO+. 4 additionally loses CO and CO2. In contrast, 2 and 3 each only exhibit a single fragmentation channel, with 2 losing C2H4 to generate protonated chloroformic acid and 3 generating protonated 2‐methylbutene by losing neutral chloroformic acid. Density functional theory at the B3LYP/6‐311+G(d,p) level of theory was employed to explore minimum energy reaction pathways for each ion, and CBS‐QB3 single‐point energy calculations were added to provide reliable energetics. The Rice–Ramsperger–Kassel–Marcus (RRKM) calculations of the rate constants for selected competing processes were carried out to link theory and experiment. One common unimolecular process observed was the 1,3‐H shift of the proton from the carbonyl oxygen to the ester oxygen before dissociation. [ABSTRACT FROM AUTHOR]

The assignment of structure by tandem mass spectrometry (MS/MS) relies on the interpretation of the fragmentation behavior of gas‐phase ions. Mass spectra were acquired for a series of heterocyclic mimetics of acidic amino acids and a related series of nitrile amino acids. All amino acids were readily protonated or deprotonated by electrospray ionization (ESI), and distinctive fragmentation processes were observed when the ions were subjected to collision‐induced dissociation (CID). The deprotonated heterocycles showed bond cleavages of the 3‐hydroxyfurazan ring with formation of oxoisocyanate and the complementary deprotonated nitrile amino acid. Further fragmentation of the deprotonated nitrile amino acids was greatly dependent on the length of the alkyl nitrile side chain. Competing losses of CO2 versus HCN occurred from α‐cyanoglycinate (shortest chain), whereas water was lost from 2‐amino‐5‐cyanopentanoate (longest chain). Interestingly, loss of acrylonitrile by a McLafferty‐type fragmentation process was detected for 2‐amino‐4‐cyanobutanoate, and several competing processes were observed for β‐cyanoalanate. In one process, cyanide ion was formed either by consecutive losses of ammonia, carbon dioxide, and acetylene or by a one‐step decarboxylative elimination. In another, complementary ions were obtained from β‐cyanoalanate by loss of acetonitrile or HN=CHCO2H. Fragmentation of the protonated 3‐hydroxyfurazan and nitrile amino acids resulted in the cumulative loss (H2O + CO), a loss that is commonly observed for protonated aliphatic α‐amino acids. Overall, the distinct fragmentation behavior of the multifunctional 3‐hydroxyfurazan amino acids correlated with the charged site, whereas fragmentations of the deprotonated nitrile amino acids showed cooperative interactions between the nitrile and the carboxylate groups. [ABSTRACT FROM AUTHOR]

Turmeric and ginger are extensively employed as functional ingredients due to their high content of curcuminoids and gingerols, considered the key bioactive compounds found in these roots. In this study, we present an innovative and fast method for the assay of curcuminoids and gingerols in different foods containing the two spices, with the aim of monitoring the quality of products from a nutraceutical perspective. The proposed approach is based on paper spray tandem mass spectrometry coupled with the use of a labeled internal standard, which has permitted to achieve the best results in terms of specificity and accuracy. All the calculated analytical parameters were satisfactory; accuracy values are around 100% for all spiked samples and the precision data result lower than 15%. The protocol was applied to several real samples, and to demonstrate its robustness and reliability, the results were compared to those arising from the common liquid chromatographic method. [ABSTRACT FROM AUTHOR]

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches. IMPORTANCE Ebola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs. [ABSTRACT FROM AUTHOR]

Long-term aging has traditionally been associated with issues such as color fading and oxidation; therefore, it limits grape wine production. Here, we analyzed 90 bottles of mulberry wine aged for various periods (up to 12 years) and observed unique trends in color, flavor, and aroma compounds during prolonged aging. Results from Somers and methylcellulose precipitation (MCP) assays indicated that the tannin and anthocyanin concentrations in newly fermented mulberry wines were 167 to 216 mg/L and 1.04 to 1.37 g/L, respectively. The total phenolics, tannins, and anthocyanin contents exhibited significant negative correlations with aging years, while the non-bleachable pigment content and hue showed positive correlations with aging times. High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis further revealed a positive correlation between the content of pyranoanthocyanins (including cyanidin-3-O-glucoside-pyruvic acid, cyanidin-3-O-glucoside-acetaldehyde, cyanidin-3-O-glucoside-4-vinocatechol, and cyanidin-3-O-glucoside-4-vinophenol) and aging times, whereas the impacts of aging on the polymeric pigment (cyanidin-3-O-glucoside-epicatechin) were not observed. This suggests that the anthocyanins in mulberry wine primarily transformed into pyranoanthocyanins rather than polymeric pigments during aging. The aging-induced reductions in protein, polysaccharide, and key aroma compounds (contributing to the fruity, sweet and floral odors) remained unaffected by prolonged aging. [ABSTRACT FROM AUTHOR]

In the early 2000s, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) emerged as a performant and relevant tool for identifying micro-organisms. Since then, it has become practically essential for identifying bacteria in microbiological diagnostic laboratories. In the last decade, it was successfully applied for arthropod identification, allowing researchers to distinguish vectors from non-vectors of infectious diseases. However, identification failures are not rare, hampering its wide use. Failure is generally attributed either to the absence of respective counter species MS spectra in the database or to the insufficient quality of query MS spectra (i.e., lower intensity and diversity of MS peaks detected). To avoid matching errors due to non-compliant spectra, the development of a strategy for detecting and excluding outlier MS profiles became compulsory. To this end, we created MSProfileR, an R package leading to a bioinformatics tool through a simple installation, integrating a control quality system of MS spectra and an analysis pipeline including peak detection and MS spectra comparisons. MSProfileR can also add metadata concerning the sample that the spectra are derived from. MSProfileR has been developed in the R environment and offers a user-friendly web interface using the R Shiny framework. It is available on Microsoft Windows as a web browser application by simple navigation using the link of the package on Github v.3.10.0. MSProfileR is therefore accessible to non-computer specialists and is freely available to the scientific community. We evaluated MSProfileR using two datasets including exclusively MS spectra from arthropods. In addition to coherent sample classification, outlier MS spectra were detected in each dataset confirming the value of MSProfileR. [ABSTRACT FROM AUTHOR]

Flavonoids are a prominent group of plant phenolics (polyphenol group) extensively present in the human diet and associated with health promotion. Flavonoids are essential in various pharmaceutical, nutraceutical, and cosmetic applications. To collect and categorize these compounds, we comprehensively assessed High-performance liquid chromatography (HPLC) techniques published to analyze flavonoids in various matrices. Most documented methods employed liquid chromatography with ultraviolet detection (HPLC-UV) and tandem mass spectrometry (LC-MS/MS) methods. Among chromatographic techniques, reversed-phase liquid chromatography remains the predominant and efficient method for separating various analytes. This review explores the primary chromatographic methods used to separate and quantify flavonoids in herbs, fruits, and biological samples. This article examines recent literature on applying liquid chromatography with different detection methods for analyzing flavonoids. To examine recent literature on applying liquid chromatography with different detection methods for analyzing flavonoids. It offers valuable insights to researchers as they prepare to develop quantification methods. [ABSTRACT FROM AUTHOR]

P0 proteins encoded by the pepper vein yellow virus (PeVYV) are pathogenic factors that cause hypersensitive response (HR). However, the host gene expression related to PeVYV P0‐induced HR has not been thoroughly studied. Transcriptomic technology was used to investigate the host pathways mediated by the PeVYV P0 protein to explore the molecular mechanisms underlying its function. We found 12,638 differentially expressed genes (DEGs); 6784 and 5854 genes were significantly upregulated and downregulated, respectively. Transcriptomic and reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR) analyses revealed that salicylic acid (SA) and jasmonic acid (JA) synthesis‐related gene expression was upregulated, and ethylene synthesis‐related gene expression was downregulated. Ultrahigh performance liquid chromatography‐tandem mass spectrometry was used to quantify SA and JA concentrations in Nicotiana benthamiana, and the P0 protein induced SA and JA biosynthesis. We then hypothesized that the pathogenic activity of the P0 protein might be owing to proteins related to host hormones in the SA and JA pathways, modulating host resistance at different times. Viral gene silencing suppression technology was used in N. benthamiana to characterize candidate proteins, and downregulating NbHERC3 (Homologous to E6‐AP carboxy‐terminus domain and regulator of choromosome condensation‐1 dmain protein 3) accelerated cell necrosis in the host. The downregulation of NbCRR reduced cell death, while that of NbBax induced necrosis and curled heart leaves. Our findings indicate that NbHERC3, NbBax, and NbCRR are involved in P0 protein‐driven cell necrosis. [ABSTRACT FROM AUTHOR]

Context: Qi‐dan‐dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. Objective: This study reveals the mechanism by which QDD ameliorates DKD. Materials and Methods: The compounds in QDD were identified by high‐performance liquid chromatography and quadrupole‐time‐of‐flight tandem mass spectrometry (HPLC‐Q‐TOF‐MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD‐low (QDD‐L), and 2% QDD‐high (QDD‐H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose‐treated HK‐2 and NRK‐52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. Results: A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (AKT) and mitogen‐activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial‐mesenchymal transition (EMT) via the p38MAPK and AKT‐mammalian target of rapamycin (mTOR) pathways. Discussion and Conclusion: QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD. [ABSTRACT FROM AUTHOR]

Pregabalin is a gabapentinoid approved for the treatment of general anxiety disorder, neuropathic pain and as adjunctive therapy for focal seizures in patients with epilepsy. In addition, there are a number of conditions for which pregabalin is prescribed off-label. Along with the widespread use there are a significant number of reports describing the misuse of pregabalin over the last decade. Over time, it became clear that pregabalin should become part of routine testing in toxicology laboratories. The aim of this paper was to present validation of a LC-MS/MS method for the quantification of pregabalin in plasma of acutely poisoned patients. Simple sample preparation step and rapid chromatographic separation shortened the overall analysis time, which was the goal of method development. The presence of pregabalin was confirmed with three ion transitions, ensuring high selectivity of the validated method. The statistical data obtained showed good precision and accuracy over a wide concentration range. No endogenous or other interference was detected, and there was no matrix effect influence with this method. The LC-MS/MS method was applied to quantify pregabalin in plasma samples of patients admitted to the emergency department due to a possible acute pregabalin overdose. Different concentrations were found, and we report, to the best of our knowledge, the highest plasma concentration of pregabalin in the plasma of a patient with acute poisoning. In conclusion, we developed a fast and simple LC-MS/MS method for reliable determination of pregabalin and demonstrated the developed method was suitable for routine use in clinical toxicology setting. [ABSTRACT FROM AUTHOR]

8-Oxoguanine (OG) is the most common oxidatively generated nucleobase damage and can mispair with adenine (A) in Hoogsteen mode during replication. Besides introducing the G·C→T·A transversion mutation, the OG·A base pair is vulnerable to ionizing radiation and one-electron oxidation owing to the lower ionization and oxidation potentials of OG than natural DNA nucleobases. Herein, we report the formation and collision-induced dissociation (CID) of the radical cation of a model base pair consisting of nucleoside-mimicking 9-methyl-8-oxoguanine (9MOG) and 9-methyladenine (9MA). The [9MOG·9MA]•+ radical cation is formed in the gas phase by redox-separation of electrospray ionization-produced CuII-nucle-obase complexes, and its CID is examined using guided-ion beam tandem mass spectrometry. Measurement included kinetic energy-dependent dissociation product ions and cross sections, from which the product pairs of [9MOG – H]• + [9MA+H]+ (major dissociation channel) and 9MOG•+ + 9MA (minor) were detected with 0 K dissociation threshold energies of 1.8 and 1. 65 eV, respectively. The [9MOG·9MA]•+ structures were examined using density functional theory, and important conformations were all featured by complete intra-base pair proton transfer as [9MOG–H]•·[9MA+H]+. On the other hand, the production of 9MOG•++9MA in dissociation required a 9MOG•+·9MA intermediate. The results were rationalized by the discovery of a double-well potential that evolves on the reaction potential energy surface of the collisionally activated base pair, leading to the proton-transfer equilibrium of excited ([9MOG–H]•·[9MA+H]+)* ⇌ (9MOG•+·9MA)*. The combined experimental and theoretical work provides insight into the less intuitive aspects of this biologically-important, non-canonical base pair, especially its opening upon oxidative and ionization damage. [ABSTRACT FROM AUTHOR]

Relative energy deficiency in sport (RED-S) is a condition that arises from persistent low energy availability (LEA), which affects the hypothalamic–pituitary axis and results in alterations of several hormones in both male and female athletes. As frequent blood hormone status determinations using venipuncture are rare in sports practice, microsampling offers promising possibilities for preventing and assessing RED-S. Therefore, this study aimed at developing a liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS/MS) method for quantifying relevant steroids and thyroid hormones in 30 μL of capillary blood obtained using Mitra® devices with volumetric absorptive microsampling technology (VAMS®). The results of the study showed that all validation criteria were met, including a storage stability of more than 28 days in a frozen state (−18 °C) and 14 days at room temperature (20 °C). The validated assay provided precise (<12%) and accurate (<13%) results for all the target analytes. Furthermore, as a proof of concept, autonomously collected VAMS® samples from 50 female and male, healthy, active adults were analyzed. The sensitivity of all analytes was adequate to quantify the decreased hormone concentrations in the RED-S state, as all authentic samples could be measured accordingly. These findings suggest that self-collected VAMS® samples offer a practical opportunity for regular hormone measurements in athletes and can be used for early RED-S assessment and progress monitoring during RED-S recovery. [ABSTRACT FROM AUTHOR]

Small molecule identification is a crucial task in analytical chemistry and life sciences. One of the most commonly used technologies to elucidate small molecule structures is mass spectrometry. Spectral library search of product ion spectra (MS/MS) is a popular strategy to identify or find structural analogues. This approach relies on the assumption that spectral similarity and structural similarity are correlated. However, popular spectral similarity measures, usually calculated based on identical fragment matches between the MS/MS spectra, do not always accurately reflect the structural similarity. In this study, we propose TransExION, a Transformer based Explainable similarity metric for IONS. TransExION detects related fragments between MS/MS spectra through their mass difference and uses these to estimate spectral similarity. These related fragments can be nearly identical, but can also share a substructure. TransExION also provides a post-hoc explanation of its estimation, which can be used to support scientists in evaluating the spectral library search results and thus in structure elucidation of unknown molecules. Our model has a Transformer based architecture and it is trained on the data derived from GNPS MS/MS libraries. The experimental results show that it improves existing spectral similarity measures in searching and interpreting structural analogues as well as in molecular networking. Scientific Contribution: We propose a transformer-based spectral similarity metrics that improves the comparison of small molecule tandem mass spectra. We provide a post hoc explanation that can serve as a good starting point for unknown spectra annotation based on database spectra. [ABSTRACT FROM AUTHOR]

Background: Despite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis. Methods: To inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively. Results: Inducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo. Conclusions: Reduced metastasis upon silencing supports EDI3's potential as a treatment target in metastasizing ER-HER2+ breast cancer. [ABSTRACT FROM AUTHOR]

Bats are the second largest mammalian order and are an endangered species group with a strong need for contamination monitoring. To facilitate non-invasive monitoring of the ecological burden in bat populations, a multiresidue method for the simultaneous quantification of 119 analytes including pesticides, persistent organic pollutants (POPs), active pharmaceutical ingredients (APIs), polycyclic aromatic hydrocarbons (PAHs), UV blockers, plasticizers, and other emerging pollutants in bat guano with gas chromatography tandem mass spectrometry (GC-MS/MS) was developed. Sample preparation and clean-up were performed with a modified QuEChERS approach based on DIN EN 15662. The method uses 1.00 g bat guano as sample with acetonitrile and water for liquid-liquid extraction. Phase separation is assisted by citrate-buffered salting out agent. For clean-up of the extract, primary secondary amine (PSA) was combined with graphitized carbon black (GCB). The lower limits of quantification (LLOQ) ranged between 2.5 and 250 µg kg−1. Linearity was shown in a concentration range from the respective LLOQs to 1250 µg kg−1. The median of the mean recovery was 102.4%. Precision was tested at three concentrations. Method and injection precision were adequate with a relative standard deviation (RSD) below 20%. Furthermore, the comparative analysis with LC-MS/MS demonstrated the reliability of the results and provided a valuable extension of the analytical scope. As proof of concept, three guano samples from a German nursery roost of Myotis myotis were analysed. The results show a time-dependent change in contaminant concentration, highlighting the strong need for non-invasive contamination monitoring of whole bat populations. [ABSTRACT FROM AUTHOR]

The study of peptide repertoires presented by major histocompatibility complex (MHC) molecules and the identification of potential T-cell epitopes contribute to a multitude of immunopeptidome-based treatment approaches. Epitope mapping is essential for the development of promising epitope-based approaches in vaccination as well as for innovative therapeutics for autoimmune diseases, infectious diseases, and cancer. It also plays a critical role in the immunogenicity assessment of protein therapeutics with regard to safety and efficacy concerns. The main challenge emerges from the highly polymorphic nature of the human leukocyte antigen (HLA) molecules leading to the requirement of a peptide mapping strategy for a single HLA allele. As many autoimmune diseases are linked to at least one specific antigen, we established FASTMAP, an innovative strategy to transiently co-transfect a single HLA allele combined with a diseasespecific antigen into a human cell line. This approach allows the specific identification of HLA-bound peptides using liquid chromatography--tandem mass spectrometry (LC-MS/MS). Using FASTMAP, we found a comparable spectrum of endogenous peptides presented by the most frequently expressed HLA alleles in the world's population compared to what has been described in literature. To ensure a reliable peptide mapping workflow, we combined the HLA alleles with well-known human model antigens like coagulation factor VIII, acetylcholine receptor subunit alpha, protein structures of the SARS-CoV-2 virus, and myelin basic protein. Using these model antigens, we have been able to identify a broad range of peptides that are in line with already published and in silico predicted T-cell epitopes of the specific HLA/model antigen combination. The transient co-expression of a single affinity-tagged MHC molecule combined with a disease-specific antigen in a human cell line in our FASTMAP pipeline provides the opportunity to identify potential T-cell epitopes/endogenously processed MHC-bound peptides in a very cost-effective, fast, and customizable system with high-throughput potential. [ABSTRACT FROM AUTHOR]

Diquat (DQ), paraquat (PQ), glufosinate (GLU), and glyphosate (GLYP) are commonly used herbicides that have been confirmed to be toxic to humans. Rapid and accurate measurements of these toxicants in clinical practice are beneficial for the correct diagnosis and timely treatment of herbicide-poisoned patients. The present study aimed to establish an efficient, convenient, and reliable method to achieve the simultaneous quantification of DQ, PQ, GLU, and GLYP in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without using derivatization or ion-pairing reagents. DQ, PQ, GLU, and GLYP were extracted by the rapid protein precipitation and liquid-liquid extraction method and then separated and detected by LC-MS/MS. Subsequently, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, extraction recovery, matrix effect, dilution integrity, and stability were evaluated to validate the method based on the FDA criteria. Finally, the validated method was applied to real plasma samples collected from 166 Chinese patients with herbicide poisoning. The results showed satisfactory linearity with low LOD (1 ng/mL for DQ and PQ, 5 ng/mL for GLU, and 10 ng/mL for GLYP, respectively) and low LOQ (5 ng/mL for DQ and PQ, 25 ng/mL for GLU and GLYP, respectively). In addition, the precision, accuracy, extraction recovery, and stability of the method were acceptable. The matrix effect was not observed in the analyzed samples. Moreover, the developed method was successfully applied to determine the target compounds in real plasma samples. These data provided reliable evidence for the application of this LC-MS/MS method for clinical poisoning detection. [ABSTRACT FROM AUTHOR]

Rationale: Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF) in North America. To investigate the effect of drug‐induced liver injury (DILI) on circulating bile acid (BA) profiles, serum from ALF patients and healthy controls were analyzed using a semitargeted high‐resolution mass spectrometry approach to measure BAs in their unconjugated and amidated forms and their glucuronide and sulfate conjugates. Methods: Human serum samples from 20 healthy volunteers and 34 ALF patients were combined with deuterated BAs and extracted, prior to liquid chromatography high‐resolution tandem mass spectrometry analysis. A mix of 46 standards helped assign 26 BAs in human serum by accurate mass and retention time matching. Moreover, other isomers of unconjugated and amidated BAs, as well as glucuronide and sulfate conjugates, were assigned by accurate mass filtering. In vitro incubations of standard BAs provided increased information for certain peaks of interest. Results: A total of 275 BA metabolites, with confirmed or putative assignments, were measured in human serum samples. APAP overdose significantly influenced the levels of most BAs, promoting glycine conjugation, and, to a lesser extent, taurine conjugation. When patient outcome was considered, 11 BAs were altered significantly, including multiple sulfated species. Although many of the BAs measured did not have exact structures assigned, several putatively identified BAs of interest were further characterized using in vitro incubations. Conclusion: An optimized chromatographic separation tailored to BAs of ranging polarities was combined with accurate mass measurements to investigate the effect that DILI has on their complex profiles and metabolism to a much wider extent than previously possible. The analysis of complex BA profiles enabled in‐depth analysis of the BA metabolism perturbations in ALF, including certain metabolites related to patient outcomes. [ABSTRACT FROM AUTHOR]

Rationale: Ensuring the global safety and effectiveness of agrochemicals has become imperative. An in‐depth understanding of impurity profiles of products is crucial, especially for high‐demand agrochemicals, where impurities may be more toxic and persistent than original agrochemicals. This study focuses on the detection and identification of impurities in a commercial chlorantraniliprole (CAP), an anthranilic diamide class broad‐spectrum insecticide. Methods: Commercial CAP was collected from an agrochemical supplier in India and was analyzed using a high‐performance liquid chromatography‐photodiode array (HPLC‐PDA) (Agilent 1260; wavelength, 220 nm) with a Zorbax RP SB‐C18 (250 × 4.6 mm, 5 μm) column and liquid chromatography–mass spectrometry (LC–MS) (Agilent 6545 quadrupole time of flight (Q‐TOF)) techniques to identify the impurities. The impurities were isolated by preparative HPLC using a Zorbax‐DB C18 (250 × 9.4 mm, 5 μm) column. liquid chromatography– tandem mass spectrometry (LC–MS/MS) experiments (Q‐TOF) were performed on CAP and its impurities to obtain their structural data. Results: HPLC‐PDA analysis of CAP showed four major impurities (IM‐1 to IM‐4) ranging from 0.76% to 4.1%. The positive ion electrospray ionization (ESI) mass spectra of CAP and its impurities showed dominant [M + H]+ ions in addition to [M + Na]+, [M + K]+, and [2M + Na]+ ions. High‐resolution mass spectrometry (HRMS) data provided the elemental composition of the compounds, and isotopic distribution patterns revealed the number of Cl and/or Br atoms present in them. The structures of impurities were proposed based on the LC–MS/MS) data and further confirmed by nuclear magnetic resonance (NMR) data on isolated impurities/synthesis. Conclusion: The quality and impurities of CAP, a popular insecticide, must be assessed and described for its efficacy and safety. In this study, four impurities of CAP were detected using HPLC and successfully characterized using LC‐HRMS, LC–MS/MS, and NMR data. The method is useful for verifying the purity of CAP as well as helping in the identification of its possible impurities. [ABSTRACT FROM AUTHOR]

Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits. [ABSTRACT FROM AUTHOR]

The salinilactones, volatile marine natural products secreted from Salinispora arenicola, feature a unique [3.1.0]‐lactone ring system and cytotoxic activities through a hitherto unknown mechanism. To find their molecular target, an activity‐based protein profiling with a salinilactone‐derived probe is applied that disclosed the protein disulfide‐isomerases (PDIs) as the dominant mammalian targets of salinilactones, and thioredoxin (TRX1) as secondary target. The inhibition of protein disulfide‐isomerase A1 (PDIA1) and TRX1 is confirmed by biochemical assays with recombinant proteins, showing that (1S,5R)‐salinilactone B is more potent than its (1R,5S)‐configured enantiomer. The salinilactones bound covalently to C53 and C397, the catalytically active cysteines of the isoform PDIA1 according to tandem mass spectrometry. Reactions with a model substrate demonstrated that the cyclopropyl group is opened by an attack of the thiol at C6. Fluorophore labeling experiments showed the cell permeability of a salinilactone‐BODIPY (dipyrrometheneboron difluoride) conjugate and its co‐localization with PDIs in the endoplasmic reticulum. The study is one of the first to pinpoint a molecular target for a volatile microbial natural product, and it demonstrates that salinilactones can achieve high selectivity despite their small size and intrinsic reactivity. [ABSTRACT FROM AUTHOR]

Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell‐permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal to selectively image, label, and crosslink aggregated proteome in live stressed cells. A combination of photo‐chemical, tandem mass spectrometry, and protein biochemistry characterizations reveals the complexity in photosensitizing pathways (both Type I & II), modification sites and labeling mechanisms. The diverse labeling sites and reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome and analyze its interactions. [ABSTRACT FROM AUTHOR]

Accurate measurement of endogenous steroid hormones in serum is a powerful tool for understanding the status of human adrenal diseases. Isotope dilution–liquid chromatography–tandem mass spectrometry is the most commonly used method for determination of steroids in serum. In this study, we used a laboratory-constructed quadrupole-linear ion trap (Q-LIT) tandem mass spectrometer to develop a new method for the quantification of five steroid hormones in serum. A simple and efficient pretreatment was improved, wherein the serum was extracted once by a mixed solvent of 1.0 mL of ethyl acetate and n-hexane. Using the optimized method, the regression coefficients of the calibration curves were all higher than 0.99, the limits of detection were from 0.31 to 1.25 ng mL−1, and the limits of quantification were between 1.00 and 2.50 ng mL−1. The intra-day (n = 3) relative standard deviations ranged from 4.24% to 13.79%, and the inter-day (n = 3) relative standard deviations from 3.99% to 11.43%. After internal standard correction, the relative recoveries of all steroids were from 86.08% to 106.60%. Twenty-two serum samples were analyzed by Q-LIT, which was compared with commercial triple quadrupole mass spectrometer. The results of the instruments had high consistency. Therefore, Q-LIT accurately quantified steroid hormones in human samples and may be used for clinical diagnosis and monitoring of hormone-dependent diseases. [ABSTRACT FROM AUTHOR]

Although Chronic Obstructive Pulmonary Disease (COPD) is highly prevalent, it is often underdiagnosed. One of the main characteristics of this heterogeneous disease is the presence of periods of acute clinical impairment (exacerbations). Obtaining blood biomarkers for either COPD as a chronic entity or its exacerbations (AECOPD) will be particularly useful for the clinical management of patients. However, most of the earlier studies have been characterized by potential biases derived from pre-existing hypotheses in one or more of their analysis steps: some studies have only targeted molecules already suggested by pre-existing knowledge, and others had initially carried out a blind search but later compared the detected biomarkers among well-predefined clinical groups. We hypothesized that a clinically blind cluster analysis on the results of a non-hypothesis-driven wide proteomic search would determine an unbiased grouping of patients, potentially reflecting their endotypes and/or clinical characteristics. To check this hypothesis, we included the plasma samples from 24 clinically stable COPD patients, 10 additional patients with AECOPD, and 10 healthy controls. The samples were analyzed through label-free liquid chromatography/tandem mass spectrometry. Subsequently, the Scikit-learn machine learning module and K-means were used for clustering the individuals based solely on their proteomic profiles. The obtained clusters were confronted with clinical groups only at the end of the entire procedure. Although our clusters were unable to differentiate stable COPD patients from healthy individuals, they segregated those patients with AECOPD from the patients in stable conditions (sensitivity 80%, specificity 79%, and global accuracy, 79.4%). Moreover, the proteins involved in the blind grouping process to identify AECOPD were associated with five biological processes: inflammation, humoral immune response, blood coagulation, modulation of lipid metabolism, and complement system pathways. Even though the present results merit an external validation, our results suggest that the present blinded approach may be useful to segregate AECOPD from stability in both the clinical setting and trials, favoring more personalized medicine and clinical research. [ABSTRACT FROM AUTHOR]

SUMMARY: Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co‐immunoprecipitation‐coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca2+‐binding proteins, ion transport‐related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca2+‐dependent protein kinases (CPKs) that significantly affected the expression of eATP‐responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP‐induced Ca2+ mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1‐mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen Botrytis cinerea. Our findings highlight CPK28, among other CPKs, as a modulator of P2K1‐mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity. Significance Statement: In the last two decades, extracellular ATP has become a pivotal signaling element in animal physiology and plant biology. Recognized for its role in plant growth and defense, the purinergic signal of eATP is well‐established. Despite some identified interactors of the eATP receptor P2K1, a comprehensive understanding of purinergic signaling has been hindered by the lack of a P2K1 interactome. Our study successfully generated this interactomics dataset, shedding light on eATP‐mediated signaling and revealing a key player, CPK28, a Ca2+‐dependent protein kinase. [ABSTRACT FROM AUTHOR]

A novel co-bonded octyl and pyridine silica (OPS) sorbent was prepared and applied for the solid phase extraction (SPE) of cyclopiazonic acid (CPA, a type of mycotoxin) in feed and agricultural products for the first time. A simple mixed-ligand one-pot reaction strategy was employed for OPS sorbent preparation. Nitrogen adsorption–desorption measurements, elemental analysis (EI), thermal gravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR) analysis demonstrated the successful immobilization of octyl and quaternary ammonium groups onto the surface of silica gel. The large specific surface area, high-density functional groups, and mixed-mode anion-exchange characteristics of these silica particles made them the ideal material for the efficient extraction of CPA. Additionally, the OPS sorbents displayed excellent batch-to-batch reproducibility, satisfactory reusability, and low cost. The SPE parameters were optimized to explore the ionic and hydrophobic interactions between CPA and the functional groups, and the ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-MS/MS) parameters were optimized to obtain a desirable extraction efficiency and high sensitivity to CPA. Meanwhile, the OPS sorbent presented a satisfactory extraction selectivity and low matrix effect. Under the optimized conditions, our developed CPA detection method was used to determine CPA level in rice, wheat flour, corn flour, peanut, and feed samples, exhibiting a lower detection limit, better linearity, higher sensitivity, and satisfactory extraction recovery rate than previously reported methods. Therefore, our method can be preferentially used as a method for the detection of CPA in agricultural products and feeds. [ABSTRACT FROM AUTHOR]

Maintaining balance is critical to minimizing astronauts' risk of falling and reducing injury or suit damage. Previous studies involving spacesuits have not examined the effects of the superior shift of the center of gravity (CoG) on astronauts' ability to balance. Here, the purpose of our study was to investigate the effects of CoG shift due to a simulated Extravehicular Mobility Unit (xEMU) on balance. Seventeen participants' standing balance was examined for three test configurations: unsuited, weighted with an Extravehicular Mobility Unit (xEMU) vest, and xEMU hard upper body torso (HUT). Using a Tekscan forceplate walkway, the CoG locations were determined. Balance assessments were performed to determine the limits of stability and standing balance performance during wide or tandem stances with eyes open/closed. The center of pressure (CoP) time series was examined in terms of displacement, velocity, and frequency measures. During the eyes-open wide stance, the xEMU vest significantly increased the mediolateral balance parameters, while the HUT significantly increased the total displacement (TOTEX), mean velocity (MVELO), and mean frequency (MFREQ) of the CoP. In the eyes-closed wide stance, the HUT significantly increased these parameters. In the eyes-closed tandem stance, the xEMU vest significantly decreased the parameters. The xEMU vest significantly reduced the TOTEX, MVELO, and MFREQ (improved standing balance), while the HUT decreased standing balance ability, seen with significant increases in said parameters. By quantifying CoG's effect on balance, our results form the basis for future balance and posture studies of xEMU spacesuits. [ABSTRACT FROM AUTHOR]

Structural annotation of small molecules in tandem mass spectrometry has always been a central challenge in mass spectrometry analysis, especially using a miniaturized mass spectrometer for on-site testing. Here, we propose the Transformer enabled Fragment Tree (TeFT) method, which combines various types of fragmentation tree models and a deep learning Transformer module. It is aimed to generate the specific structure of molecules de novo solely from mass spectrometry spectra. The evaluation results on different open-source databases indicated that the proposed model achieved remarkable results in that the majority of molecular structures of compounds in the test can be successfully recognized. Also, the TeFT has been validated on a miniaturized mass spectrometer with low-resolution spectra for 16 flavonoid alcohols, achieving complete structure prediction for 8 substances. Finally, TeFT confirmed the structure of the compound contained in a Chinese medicine substance called the Anweiyang capsule. These results indicate that the TeFT method is suitable for annotating fragmentation peaks with clear fragmentation rules, particularly when applied to on-site mass spectrometry with lower mass resolution. Tandem mass spectrometry spectra contain structural information of analyzed small molecules, however structural annotation and prediction from MS spectra remain challenging. Here, the authors combine the fragmentation tree models with a deep-learning transformer module, to annotate the fragmentation peaks and generate de novo molecular structures from a low-resolution mass spectrometer. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ~9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations. [ABSTRACT FROM AUTHOR]

We present an analytical method to detect and quantify residues of currently used pesticides (CUPs), which include 31 active ingredients (ai) and seven transformation products (TPs) in tropical and agricultural soils of Cuba. Ten isotopically labeled analogous compounds served as internal standards (IL-IS). The novelty of this research is the inclusion of different tropical soils type scarcely studied for CUPs and TPs, based on the QuEChERS (quick, easy, cheap, effective, rugged and safe) method, followed by chromatography tandem mass spectrometry. All figures of merit proved to be satisfactory according to SANTE guidelines 2020 and 2021. Matrix effects (ME) calculated by the external standard method were significant (|ME| > 20% for almost all compounds; grand mean ± standard deviation (STD) 104 ± 108%) in all soils. The internal standard method compensated ME to non-significant levels (8 ± 50%), even for analytes with a non-structure identical IL-IS (STD, 13 ± 57%). Repeatability (relative standard deviation, RSDr) and reproducibility (RSDR) for skeletic regosol (SR) were 7.5 ± 2.8% and 11.7 ± 4.7%, respectively. Absolute (quantified for 11 analytes with structure identical IL-IS) and relative recovery from SR was 92 ± 13% (mean ± STD) and 90 ± 12%, respectively. Limits of quantification for SR ranged from 0.1 to 10 ng/g, except metalaxyl and oxyfluorfen (25 ng/g each). Linearity of matrix-matched (MM) calibration curves (5 to 100 ng/g) had an R2 of ≥ 0.99 for all soils and almost all analytes. The method was successfully applied to 30 real soil samples. [ABSTRACT FROM AUTHOR]

Purpose: The Discovery of underlying intermediates associated with the development of dyslipidemia results in a better understanding of pathophysiology of dyslipidemia and their modification will be a promising preventive and therapeutic strategy for the management of dyslipidemia. Methods: The entire dataset was selected from the Surveillance of Risk Factors of Noncommunicable Diseases (NCDs) in 30 provinces of Iran (STEPs 2016 Country report in Iran) that included 1200 subjects and was stratified into four binary classes with normal and abnormal cases based on their levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and non-HDL-C. Plasma concentrations of 20 amino acids and 30 acylcarnitines in each class of dyslipidemia were evaluated using Tandem mass spectrometry. Then, these attributes, along with baseline characteristics data, were used to check whether machine learning (ML) algorithms could classify cases and controls. Results: Our ML framework accurately predicts TG binary classes. Among the models tested, the SVM model stood out, performing slightly better with an AUC of 0.81 and a standard deviation of test accuracy at 0.04. Consequently, it was chosen as the optimal model for TG classification. Moreover, the findings showed that alanine, phenylalanine, methionine, C3, C14:2, and C16 had great power in differentiating patients with high TG from normal TG controls. Conclusions: The comprehensive output of this work, along with sex-specific attributes, will improve our understanding of the underlying intermediates involved in dyslipidemia. [ABSTRACT FROM AUTHOR]

A pinanamine‐based analog, (1R,2R,3R,5S)‐N‐((3‐cyclopropylthiophen‐2‐yl)methyl)‐2,6,6‐trimethylbicyclo[3.1.1]heptan‐3‐amine (M090) was synthesized and demonstrated with anti‐influenza activity to overcome the multi‐drug resistance. Herein, a rapid and robust liquid chromatography‐tandem mass spectrometry method was developed and validated to quantify M090 in rat plasma. With simple protein precipitation by methanol, the separation of M090 was performed on a Waters BEH‐C18 column (2.1 × 100 mm, 1.7 μm) by gradient elution at 0.4 mL/min with mobile phases consisting of water containing 0.1 % formic acid (phase A) and methanol (phase B). M090 and clenbuterol (internal standard) were detected using multiple reaction monitoring in positive electrospray ionization mode with transitions of m/z 290.3→137.0 and m/z 277.0→203.0, respectively. The method was validated over a linear range of 1.0–2400 ng/mL with a regression coefficient of 0.9974 and no endogenous interference. The intra‐ and inter‐batch precisions were within 8.29% and accuracy ranged from 100.3% to 108.1% for M090. The validated method was utilized to evaluate the in vitro metabolic stability and in vivo pharmacokinetics of M090 in rats. [ABSTRACT FROM AUTHOR]

Herbicide residue levels were analyzed in agricultural soils of Batak plain and health risk assessments were made for relevant pesticides. Herbicide contamination levels were analyzed with the use of Quick-Easy-Cheap-Efficient-Rugged-Safe (QuEChERS)-liquid chromatography/tandem mass spectrometry (LC–MS/MS) procedure. Herbicide-free soil samples were spiked at two different levels. Overall recovery of the method was 87.32%. Present findings were parallel to SANTE recovery limits. About 50% of collected samples from the study sites contained herbicides at different concentrations. Totally, eight herbicides were detected, and herbicide concentrations ranged between 1.085 and 1724.23 μg kg−1. Metolachlor had the highest concentration (1724.23 μg kg−1) in a sample taken close to the pesticide waste disposal area. Six herbicides were detected at different concentrations in the same sample. Persistent herbicides (terbuthylazine and pendimethalin) were detected in 35 samples. Risk assessments revealed that hazard index (HI) and hazard quotient (HQ) were less than 1. The greatest HQ values were identified for terbuthylazine as 2772.48 × 10−7 and 20793.61 × 10−7 for adults and children, respectively. The HI for all herbicides were 3916.05 × 10−7 for adult and 29370.39 × 10−7 for children. [ABSTRACT FROM AUTHOR]

Regular monitoring of pesticide residues in potatoes (Solanum tuberosum L.) is important to ensure a low risk of pesticide toxicity to consumers. An efficient, modified quick easy cheap effective rugged safe‐dispersive‐solid‐phase extraction (QuEChERS‐ d‐SPE) was optimized and validated for quantification of pesticide residues (metalaxyl, chlorpyrifos, λ‐cyhalothrin, and cypermethrin) in popular Kenyan potato varieties "Shangi," "Tigoni," and "Dutch Robyjn," grown under controlled field conditions using gas chromatography with tandem mass spectrometry (GC‐MS/MS). An evaluation of the effect of five spraying frequencies (unsprayed, 5, 7, 10, and 14‐day intervals) of metalaxyl‐based fungicides on residue levels in harvested potatoes was simultaneously done. Metalaxyl residue levels in the varieties were determined for each spraying regime. Method validation showed good linearity (r2) of 0.988–0.999, detection limit (0.37–0.61 µg/L), quantification limit (1.20–1.84 µg/L) while recovery (71%–94%), and precision (relative standard deviation of 2.3%–24.1%) were used to determine the accuracy of the method. Pyrethroids (cypermethrin and λ‐cyhalothrin) and organophosphates (metalaxyl and chlorpyrifos) were detected in the potato samples. Residue levels of the compounds were not significantly different (p = 0.05) between varieties except for chlorpyrifos. Cypermethrin was the residue compound detected at the highest mean level with 17.4 µg/L, followed by metalaxyl, chlorpyrifos, and λ‐cyhalothrin, at 2.88, 1.05, and 0.15 µg/L, respectively. Residue levels at different spraying frequencies varied statistically between varieties (p = 0.05). Dutch Robyjn sprayed at seven days' intervals had the highest residue level of 9.49 ± 0.26 µg/L. QuEChERS‐d‐SPE with GC‐MS/MS has shown suitability for routine pesticide residue monitoring in potatoes. The potatoes grown under the controlled experimental conditions did not present any concern for human health, as the pesticide residues detected were below Codex maximum residue levels of 500 µg/L. Core Ideas: Potato field production in Kenya is characterized by intensive pesticide application.There is limited data available locally about the type of pesticide or their residue levels present in potatoes.Pyrethroids and organophosphates were detected and quantified in the potato samples.Cypermethrin was the highest residue compound detected, though below Codex MRLs while λ‐cyhalothrin was the lowest.Highest residue in cypermethrin in Dutch Robyjn, metalaxyl in Shangi, and chlorpyrifos and λ‐cyhalothrin in Tigoni. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase. [ABSTRACT FROM AUTHOR]

Proteomics has been used extensively in the field of mycology, mainly in trying to understand the complex network of protein-protein interactions that has been implicated in the molecular functions of fungi. It is also a useful tool to compare metabolic differences within a genus. Species of Pseudogymnoascus, a genus under the phyla Ascomycota, have been shown to play an important role in the soil environment. They have been found in both polar and temperate regions and are a known producer of many extracellular hydrolases that contribute to soil decomposition. Despite the apparent importance of Pseudogymnoascus spp. in the soil ecosystem, investigations into their molecular functions are still very limited. In the present study, proteomic characterization of six Pseudogymnoascus spp. isolated from three biogeographic regions (the Arctic, Antarctic, and temperate regions) was carried out using tandem mass spectrometry. Prior to proteomic analysis, the optimization for protein extraction was carried out. Trichloroacetic acid‑acetone‑phenol was found to be the best extraction method to be used for proteomic profiling of Pseudogymnoascus spp. The proteomic analysis identified 2003 proteins that were successfully mapped to the UniProtKB database. The identified proteins were clustered according to their biological processes and molecular functions. The shared proteins found in all Pseudogymnoascus spp. (1201 proteins) showed a significantly close relationship in their basic cellular functions, despite differences in morphological structures. Analysis of Pseudogymnoascus spp. proteome also identified proteins that were unique to each region. However, a high number of these proteins belonged to protein families of similar molecular functions, namely, transferases and hydrolases. Our proteomic data can be used as a reference for Pseudogymnoascus spp. across different global regions and a foundation for future soil ecosystem function research. [ABSTRACT FROM AUTHOR]

Introduction: Docetaxel, a taxane used in the treatment of solid tumours, exerts pharmacological activity when in its unbound form. We report a sensitive assay to quantify unbound docetaxel after oral administration of docetaxel plus encequidar (oDox+E). Unbound drug quantification is important due to its direct correlation with drug-related toxicity and therapeutic efficacy. We improve on the sensitivity of current assay methods and demonstrate the utility of the assay on a novel formulation of oral docetaxel. Methods: Ultrafiltration followed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) was utilized. Long-term stability, precision, accuracy, and recovery experiments were conducted to validate the assay. Additionally, patient samples from a Phase I dose-escalation pharmacokinetic study were analyzed using the developed assay. Results: The assay method exhibited long-term stability with an observed change between 0.8 and 6.9% after 131 days of storage at −60 °C. Precision and accuracy quality controls met the FDA acceptance criteria. An average recovery of 88% was obtained. Patient sample analysis demonstrated successful implementation of the assay. Conclusion: A validated sensitive assay was developed with an LLOQ of 0.084 ng/mL using 485 µL of human plasma. The sensitivity of the assay allowed quantification of unbound docetaxel concentrations in an early-phase oDox+E clinical study to compare it against IV docetaxel using pharmacokinetic modelling. Successful development of oDox+E represents an opportunity to replace the current IV docetaxel regimen with an oral regimen with lower cost, decreased side effects, and improve patient quality of life and experience. [ABSTRACT FROM AUTHOR]

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology. We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction. DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography—Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest. [ABSTRACT FROM AUTHOR]

The aim of this study is to develop a rapid and accurate method for simultaneous analysis of multi-residue pesticides and conduct pesticide monitoring in agricultural products produced by the production and distribution stage in Korea. The representative agricultural products were selected as brown rice, soybean, potato, mandarin, and green pepper and developed using gas chromatography with tandem mass (GC-MS/MS) for the analysis of 272 pesticide residues. The experimental samples were extracted by the QuEChERS-EN method and then cleaned up by using d-SPE, including MgSO4 and primary secondary amine (PSA) sorbents. The established method was validated in accordance with Codex CAC-GL/40, and the limit of quantitation (LOQ) was determined to be 0.01 mg/kg. A total of 243 pesticides satisfied the guidelines in five samples at three levels with values of 60 to 120% (recovery) and ≤45% (coefficient of variation, CV). The remaining 29 pesticides did not satisfy the guidelines, and these pesticides are expected to be used as a screening method for the routine inspection of agricultural products. As a result of analyzing 223 agricultural products in South Korea by applying the simultaneous analysis method, none of the detected levels in the samples exceeded the standard values based on maximum residue limits (MRLs). The developed method in this study will be used to inspect residual pesticides in agricultural products, and it is anticipated to contribute to the distribution of safe agricultural products to consumers. [ABSTRACT FROM AUTHOR]

Flavonoids are important secondary metabolites found in Juglans mandshurica Maxim., which is a precious reservoir of bioactive substances in China. To explore the antitumor actions of flavonoids (JMFs) from the waste branches of J. mandshurica, the following optimized purification parameters of JMFs by macroporous resins were first obtained. The loading concentration, flow rate, and loading volume of raw flavonoid extracts were 1.4 mg/mL, 2.4 BV/h, and 5 BV, respectively, and for desorption, 60% ethanol (4 BV) was selected to elute JMFs-loaded AB-8 resin at a flow rate of 2.4 BV/h. This adsorption behavior can be explained by the pseudo-second-order kinetic model and Langmuir isotherm model. Subsequently, JMFs were identified using Fourier transform infrared combined with high-performance liquid chromatography and tandem mass spectrometry, and a total of 156 flavonoids were identified. Furthermore, the inhibitory potential of JMFs on the proliferation, migration, and invasion of HepG2 cells was demonstrated. The results also show that exposure to JMFs induced apoptotic cell death, which might be associated with extrinsic and intrinsic pathways. Additionally, flow cytometry detection found that JMFs exposure triggered S phase arrest and the generation of reactive oxygen species in HepG2 cells. These findings suggest that the JMFs purified in this study represent great potential for the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

• We applied liquid chromatography-tandem mass spectrometry to the analysis of historical iron gall ink recipes. • We characterised ageing markers of iron gall inks by HPLC-ESI-Q-ToF. • We highlighted different degradation trends depending on historical recipes and ageing conditions. • We proved that it is possible to use 15 µg as minimum amount of sample for the HPLC-ESI-Q-ToF analysis of iron gall inks. Several manuscripts and drawings of our historical and artistic heritage have been produced with iron gall inks. To obtain an iron gall ink, ancient treatises cite the addition of ferrous sulphate and gum Arabic to a decoction of oak galls as a basic procedure. Owing to the development of synthetic chemistry, iron gall ink recipes were improved with new materials and procedures in the late 19th and early 20th century. Notably, many conservation issues arise from the interaction between iron gall inks and the paper support of manuscripts and drawings. To date, most of the research on the topic are focused on paper preservation by non-destructive analytical methods, which provide only limited information on degradation process trends and minor components, representative of iron gall ink's recipes. In the present work, three historical recipes of iron gall inks (alizarine ink, Reid ink, modern gall ink) , dated to 19th-20th century and differing for the preparation method and additives, were characterized. The molecular markers of iron gall inks and of gallic acid degradation were detected by an optimized protocol based on high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC HRMS). Furthermore, by performing ageing tests on reference materials in different indoor conditions (natural light and stored in the dark), two degradation mechanisms were observed: hydrolysis of poly-galloyl glucose species and auto-oxidation of gallic acid. Thus, different chemical profiles and ageing trends were revealed depending on the starting recipe. The procedure Limit of Detection (LOD) was estimated, improving the approaches reported so far in the literature. Finally, the strategy was successfully applied for the characterisation of the ink employed in a very degraded 16th century manuscript, granting access to the complete molecular profile of an iron gall ink with just 15 µg of sample. [Display omitted] [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) analysis has become crucial due to their presence in the environment, their persistence and potential health risks. These compounds are commonly used in food contact materials (FCM) as a coating to provide water and grease-repellent properties. One of the pathways for PFAS to enter the human body is either through direct consumption of contaminated food or indirectly through migration from FCM into food. The purpose of this study was to investigate where the initial contamination of paper FCM occurs. We analysed paper material consisting of fresh fibre and secondary materials, intended to produce food packaging for the presence of PFAS. The samples were extracted and analysed for 23 different PFAS substances using the targeted approach with LC tandem mass spectrometry (LC-MS/MS). This analytical technique detects specific, easily ionisable PFAS with high sensitivity. However, one drawback of this approach is that it allows the identification of less than 1% of the PFAS known today. For this reason, we used combustion ion chromatography (CIC) to determine the content of extractable organic fluorine compounds (EOF) and compare it to the total fluorine content. The targeted analysis using LC-MS/MS measured an average sum concentration of PFAS of 0.17 ng g−1 sample. Our research shows that the primary PFAS contamination happens during the recycling process since all of the samples in which the targeted PFAS were measured belonged to the secondary material. The most frequently detected analytes were PFOA and PFOS, detected in 90% and 62% of the samples, respectively, followed by PFBS (in 29% of the samples). CIC showed that measured PFAS via LC-MS/MS amount to an average of 2.7 × 10−4% of total fluorine content, whereas the EOF was under the LOD in all of the measured samples. This result highlights the complexity of the accurate determination of PFAS compounds, displaying what kind of information the chosen methods provide. [ABSTRACT FROM AUTHOR]

To investigate cannabinoid content and profiles, 16 cannabinoids were quantified in 30 commercial hemp seed edible oils. In addition, one hemp seed oil was subjected to thermal processing up to 200 °C for up to 60 min. UHPLC-MS/MS was used for analysis. The content of cannabinoids in the samples ranged from 9 to 279 mg kg−1 (sum) and for Δ9-tetrahydrocannabinol (Δ9-THC) from 0.2 to 6.7 mg kg−1. Three samples exceeded the EU Δ9-THC equivalent maximum levels of 7.5 mg kg−1 for hemp seed oils. Cannabinoid profiles can provide indications of different product characteristics (e.g. degree of processing, variety of plant material). Furthermore, intense thermal processing (200 °C, 60 min) led to 38% decrease in sum cannabinoid content (sum of all analysed cannabinoids in this study), 99% decrease in cannabinoid acids, and 22% increase in Δ9-THC. [ABSTRACT FROM AUTHOR]

BACKGROUND: It is reported that anti‐enterovirus 71 (EV71) drugs have some side effects on human health. Notably, fungi plays a crucial role in promoting human health and anti‐virus. Grifola frondosa is a type of large medicinal and edible fungi, rich in active substances. The present study aimed to investigate the anti‐EV71 effect of G. frondosa and the potential active substances. RESULTS: In the present study, the water extract of G. frondosa was subjected to ethanol precipitation to obtain the water‐extracted supernatant of G. frondosa (GFWS) and water‐extracted precipitation of G. frondosa. Their inhibitory effects on EV71 virus were studied based on a cell model. The results showed that GFWS had stronger security and anti‐EV71 effects. In addition, the chemical constituents of GFWS were identified by ultra‐high performance liquid chromatography‐tandem mass spectrometry, which were selected for further separation and purification. Three compounds, N‐butylaniline, succinic acid and l‐tryptophan, were isolated from GFWS by NMR spectroscopy. It is noteworthy that N‐butylaniline and l‐tryptophan were isolated and identified from the G. frondosa fruiting bodies for the first time. Our study found that l‐tryptophan has anti‐EV71 virus activity, which reduced EV71‐induced apoptosis and significantly inhibited the replication process after virus adsorption. Furthermore, it could also bind to capsid protein VP1 to prevent the virus from attaching to the cells. CONCLUSION: l‐tryptophan was an inhibitor of the EV71 virus, which could be used in infant nutrition and possibly provide a new drug to treat hand, foot and mouth disease. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Context Reliable estradiol (E2) reference intervals (RIs) are crucial in pediatric endocrinology. Objectives This study aims to develop a sensitive ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method for E2 in serum, to establish graphically represented RI percentiles and annual RIs for both sexes, and to perform a systematic literature comparison. Methods First, a UPLC-MS/MS method for E2 was developed. Second, graphically represented RI percentiles and annual RIs covering 0-18 years were computed (cohort of healthy children [1181 girls and 543 boys]). Subsequently, RIs were compared with published data by systematic searches. Results Lower limit of quantification was 11 pmol/L, indicating high sensitivity. Estradiol first peaked during mini-puberty in both sexes (girls up to 192 pmol/L; boys up to 225 pmol/L). As could be expected, girls showed higher pubertal E2 (up to 638 pmol/L). However, boys' RIs (up to 259 pmol/L) overlapped considerably. We found 4 studies in the literature that also used LC-MS/MS to determine E2 and published RIs for the complete pediatric age range. Reference intervals varied considerably. Pre-pubertal and pubertal phases were present in all studies. Higher E2 during the time of mini-puberty in both sexes was documented in 3 studies including ours. Conclusions Variability of RIs for E2 between studies illustrates the importance of laboratory-specific RIs despite using a LC-MS/MS reference method. In boys, the striking E2 peak during mini-puberty as well as high pubertal E2 without phenotypic estrogenization in regular male puberty indicates that the role of E2 in children and, especially in boys, requires better functional understanding. [ABSTRACT FROM AUTHOR]

Newborn screening (NBS) for sickle cell disease (SCD) requires a robust, high-throughput method to detect hemoglobin S (HbS). Screening for SCD is performed by qualitative methods, such as isoelectric focusing (IEF), and both qualitative and quantitative methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and tandem mass spectrometry (MS/MS). All these methods detect HbS, as well as low-level or absent HbA, and also other variants of hemoglobin. HPLC is considered as a reference method for NBS, because of its high sensitivity and specificity in detecting HbS. NeoSickle®, a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, combined with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation, has increased the throughput of SCD testing. The purpose of this study was to compare the performances of NeoSickle® and HPLC. A prospective study was conducted including 9,571 samples from the NBS program to compare MALDI-MS using NeoSickle® with an HPLC method. Correlation between the two methods was studied. For the MALDI-MS method, sensitivity, specificity, NPV, and PPV were calculated. We found over 99.4 % correlation between the HPLC and MALDI-MS results. NeoSickle® showed 100 % of sensitivity and specificity in detecting SCD syndrome, leading to positive and negative predictive values of 100 %. NeoSickle® is adapted to NBS for SCD, and can be used in first-line high-throughput screening to detect HbS, and beta-thalassemia major warning. When HbS is detected, second-line use of another specific method as HPLC is necessary. [ABSTRACT FROM AUTHOR]

Energy‐resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI‐MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo‐multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 μM up to 2.5 μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples. [ABSTRACT FROM AUTHOR]

The profile of secondary metabolites present in the apple cuticular layer is not only characteristic of a particular apple cultivar; it also dynamically reflects various external factors in the growing environment. In this study, the possibility of authenticating apple samples by analyzing their cuticular layer extracts was investigated. Ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for obtaining metabolomic fingerprints. A total of 274 authentic apple samples from four cultivars harvested in the Czech Republic and Poland between 2020 and 2022 were analyzed. The complex data generated, processed using univariate and multivariate statistical methods, enabled the building of classification models to distinguish apple cultivars as well as their geographical origin. The models showed very good performance in discriminating Czech and Polish samples for three out of four cultivars: "Gala", "Golden Delicious" and "Idared". Moreover, the validity of the models was tested over several harvest seasons. In addition to metabolites of the triterpene biosynthetic pathway, the diagnostic markers were mainly wax esters. "Jonagold", which is known to be susceptible to mutations, was the only cultivar for which an unambiguous classification of geographical origin was not possible. [ABSTRACT FROM AUTHOR]

Objectives: The differences in chemical constituents in different parts of Tetrastigma hemsleyanum were analyzed. Methods: The contents of total phenols, total flavonoids, and polysaccharides in methanol extracts from different parts of T. hemsleyanum in Zhejiang were determined, and the chemical components of different parts of T. hemsleyanum were collected by ultra-performance liquid chromatography-tandem quadrupole-time-of-flight mass spectrometry. The differential index components of different parts of T. hemsleyanum were identified and analyzed by accurate mass-to-charge ratio of primary mass spectrometry and fragment ion of secondary mass spectrometry with reference spectrum and software database search and related literature reports. Results: A total of 20 components were identified, including 13 identified in root tubers, 7 in fibrous roots and 8 in leaves, and the structural cleavage of its compounds was analyzed. Conclusion: It provides a possibility and theoretical basis for the future use of the whole plant of T. hemsleyanum as medicine. [ABSTRACT FROM AUTHOR]