Progressive expansion of albumin adducts for organophosphorus nerve agent traceability based on single and group adduct collection.

Protein adducts are important biological targets for traceability of organophosphorus nerve agents (OPNAs). Currently, the recognized biomarkers that can be used in actual samples in the field of chemical forensics only include Y411 in albumin and the active nonapeptide in butyrylcholinesterase (BChE). To explore stable and reliable protein adducts and increase the accuracy of OPNAs traceability further, we gradually expanded OPNAs-albumin adducts based on single and group adduct collection. Several stable peptides were found via LC-MS/MS analysis in human serum albumin (HSA) exposed to OPNAs in a large exposure range. These adducts were present in HSA samples exposed to OPNAs of each concentration, which provided data support for the reliability and stability of using adducts to trace OPNAs. Meanwhile, the formation mechanism of OPNAs-cysteine adduct was clarified via computer simulations. Then, these active sites found and modified peptides were used as raw materials for progressive expansion of albumin adducts. We constructed an OPNAs-HSA adducts group, in which a specific agent is the exposure source, and three or more active peptides constitute data sets for OPNAs traceability. Compared with single or scattered protein adducts, the OPNAs-HSA adduct group improves OPNAs identification by mutual verification using active peptides or by narrowing the identity range of the exposure source. We also determined the minimum detectable concentration of OPNAs for the adduct group. Two or more peptides can be detected when there is an exposure of 50 times the molar excess of OPNAs in relation to HSA. This improved the accuracy of OPNAs exposure and identity confirmation. A collection of OPNAs-albumin adducts was also examined. The collection was established by collecting, classifying, and integrating the existing albumin adducts according to the species to which each albumin belongs, the types of agents, and protease. This method can serve as a reference for discovering new albumin adducts, characteristic phosphonylated peptides, and potential biomarkers. In addition, to avoid a false negative for OPNAs traceability using albumin adducts, we explored OPNAs-cholinesterase adducts because cholinesterase is more reactive with OPNAs than albumin. Seven active peptides in red blood cell acetylcholinesterase (RBC AChE) and serum BChE can assist in OPNAs exposure and identity confirmation.

Study on phosphonylation and modification characteristics of organophosphorus nerve agents on multi-species and multi-source albumins.

Protein adducts are vital targets for exploring organophosphorus nerve agents (OPNAs) exposure and identification, that can be used to characterize the chemical burden and initiate chemical safety measures. However, the use of protein adducts as biomarkers of OPNA exposure has developed slowly. To further promote the development of biomarkers in chemical forensics, it is crucial to expand the range of modified peptides and active sites, and describe the characteristics of OPNA adducts at specific reaction sites. This study utilized multi-species and multi-source albumins as the protein targets. We identified 56 peptides in albumins from various species (including human, horse, rat and pig), that were modified by at least two OPNAs. Diverse modification characteristics were observed in response to certain agents: including (1) multiple sites on the same peptide modified by one or more agents, (2) different reactivities at the same site in homologous albumins, and (3) different preferences at the same active sites associated with differences in the biological matrix during exposure. Our studies provided an empirical reference with rationalized underpinnings supported by estimated conformation energetics through molecular modeling. We employed different peptide markers for detection of protein adducts, as (one would do) in forensic screening for identification and quantification of chemical damage. Three characteristic peptides were screened and analyzed in human albumin, including Y287ICENQDSISSK, K438VPQVS443TPTLVEVSR, and Y162LY164EIAR. Stable fragment ions with neutral loss were found from their tandem MS/MS spectra, which were used as characteristic ions for identification and extraction of modified peptides in enzymatic digestion mixtures. Coupling these observations with computer simulations, we found that the structural stability of albumin and albumin-adduct complexes (as well as the effective force that promotes stability of different adducts) changes in the interval before and after adduct formation. In pig albumin, five active peptides existed stably in vivo and in vitro. Most of them can be detected within 30 min after OPNA exposure, and the detection window can persist about half a month. These early findings provided the foundation and rationale for utilizing pig albumin as a sampling target for rapid analysis in future forensic work.

Application of chemical attribution in matching OPNAs-exposed biological samples with exposure sources- based on the impurity profiles via GC × GC-TOFMS analysis.

Chemical attribution is a vital tool to attribute chemicals or related materials to their origins in chemical forensics via various chemometric methods. Current progress related to organophosphorus nerve agents (OPNAs) has mainly focused on the attribution of chemical sources and synthetic pathways. It has not yet been applied in matching exposed biological samples to their sources. This work used chemical attribution to explore organic impurity profiles in biological samples exposed to various OPNAs. Chemical attribution was first used to identify the exposure source of biological samples based on the full-scan data via comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometer (GC × GC-TOFMS). Taking peak area as the only variable, it can quickly match exposed samples to their sources by applying unsupervised or supervised models, screen difference compounds via one-way ANOVA or t-tests, and then identify valuable impurities that can distinguish different types of exposed samples. To further obtain the impurity profile only applicable to a certain weapon' samples, the irrelevant components were removed via conventional methods. The findings showed there were 53 impurities that can promote distinguishing six groups of OPNA exposed samples, as well as 42 components that can be used as valuable impurities to distinguish class G and class V samples. These were all unique impurities that appear in a certain weapon' samples. The outcomes can be a reference for tracing the source for OPNA-exposed samples, which was beneficial to the further development in source matching of forensic samples. Moreover, the chemical attribution for impurity profiles in biological samples after weapons exposure may inspire research into the characteristics of impurity profile in biological samples as well as practical applications of chemical attribution for OPNA-exposed samples, that may expand potential biomarkers and break the limits of existing markers in the future.

Synthesis, biological evaluation and molecular docking of novel nereistoxin derivatives containing phosphonates as insecticidal/AChE inhibitory agents.

In continuation of our program aimed at the discovery and development of natural product-based insecticidal agents, a series of novel nereistoxin derivatives containing phosphonate were synthesized and characterized by 31P, 1H, 13C NMR and HRMS. The bioactivities of the derivatives were evaluated for the acetylcholinesterase (AChE) inhibition potency and insecticidal activity. The AChE inhibitory effects of the derivatives were investigated using the in vitro Ellman method. Half of the compounds exhibited excellent inhibition of AChE. All the compounds were assessed for insecticidal activities against Mythimna separate (Walker) and Rhopalosiphum padi in vivo. Some derivatives displayed promising insecticidal activity against Rhopalosiphum padi. Compounds 5b and 6a displayed the highest activity against R. padi, showing LC50 values of 17.14 and 18.28 µg mL-1, respectively, close to that of commercial insecticide flunicotamid (LC50 = 17.13 µg mL-1). Compound 9g also showed notable insecticidal activity, with an LC50 value of 23.98 µg mL-1. Additionally, the binding modes of the active compounds 5b, 6a and 9g with AChE were analyzed in-depth though molecular docking and the intrinsic reasons for the differences in the strength of the compound's activities were elucidated. In summary, our findings demonstrate the potential of these nereistoxin derivatives as promising candidates for the development of novel pesticides.

Synthesis, Bioactivity and Molecular Docking of Nereistoxin Derivatives Containing Phosphonate.

Novel nereistoxin derivatives containing phosphonate were synthesized and characterized via 31P, 1H and 13C NMR and HRMS. The anticholinesterase activity of the synthesized compounds was evaluated on human acetylcholinesterase (AChE) using the in vitro Ellman method. Most of the compounds exhibited good inhibition of acetylcholinesterase. All of these compounds were selected to assess their insecticidal activity (in vivo) against Mythimna separata Walker, Myzus persicae Sulzer and Rhopalosiphum padi. Most of the tested compounds displayed potent insecticidal activity against these three species. Compound 7f displayed good activity against all three insect species, showing LC50 values of 136.86 µg/mL for M. separata, 138.37 µg/mL for M. persicae and 131.64 µg/mL for R. padi. Compound 7b had the highest activity against M. persicae and R. padi, with LC50 values of 42.93 µg/mL and 58.19 µg/mL, respectively. Docking studies were performed to speculate the possible binding sites of the compounds and explain the reasons for the activity of the compounds. The results showed that the compounds had lower binding energies with AChE than with the acetylcholine receptor (AchR), suggesting that compounds are more easily bound with AChE.

Chemometrics-assisted analysis of chemical impurity profiles of tabun nerve agent using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

A route determination strategy through non-targeted screening of chemical attribution signatures was developed to identify tabun samples from three different synthetic routes. The CAS profiles of tabun samples were obtained by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometer (GC×GC-TOFMS). The structures of 109 CAS were identified by NIST library and mass spectrometry fragment analysis. A few identified compounds could be traced to impurities in precursor compounds used in tabun synthesis. Based on the gas chromatography/mass spectrometry peak data of the selected CAS, partial least squares-discriminant analysis (PLS-DA) was used to extract the chemical attribution signatures of the characteristic compounds. The trained PLS-DA model performed well, with the lowest specificity and sensitivity values of 1.000 and 0.882, respectively. The performance of the PLS-DA model was further verified by unknown sample test set. The model demonstrated its ability to differentiate all the unknown tabun samples. The stability of chemical attribution signatures from different time periods was also investigated, and was further evaluated.

Current Progress for Retrospective Identification of Nerve Agent Biomarkers in Biological Samples after Exposure.

Organophosphorus neurotoxic agents (OPNAs) seriously damage the nervous system, inhibiting AChE activity and threatening human health and life. Timely and accurate detection of biomarkers in biomedical samples is an important means for identifying OPNA exposure, helping to recognize and clarify its characteristics and providing unambiguous forensic evidence for retrospective research. It is therefore necessary to summarize the varieties of biomarkers, recognize their various characteristics, and understand the principal research methods for these biomarkers in the retrospective detection of OPNA exposure. Common biomarkers include mainly intact agents, degradation products and protein adducts. Direct agent identification in basic experimental research was successfully applied to the detection of free OPNAs, however, this method is not applicable to actual biomedical samples because the high reactivity of OPNAs promotes rapid metabolism. Stepwise degradation products are important targets for retrospective research and are usually analyzed using a GC-MS, or an LC-MS system after derivatization. The smaller window of detection time requires that sampling be accomplished within 48 h, increasing the obstacles to determining OPNA exposure. For this reason, the focus of retrospective identification of OPNA exposure has shifted to protein adducts with a longer lifetime. Compared to the fluoride-induced reactivation method, which cannot be used for aged adducts, digestive peptide analysis is the more elegant method for detecting various adducts, identifying more active sites, exploring potential biomarkers and excavating characteristic ions. Retrospective identification of biomarkers after OPNA poisoning is of primary importance, providing unambiguous evidence for forensic analysis in actual cases and judgment of chemical accidents. At present, degradation products, the nonapeptide from BChE adducts and Y411 from human serum adducts are used successfully in actual cases of OPNA exposure. However, more potential biomarkers are still in the discovery stage, which may prove inconclusive. Therefore, there is an urgent need for research that screens biomarker candidates with high reactivity and good reliability from the potential candidates. In addition, mass spectrometry detection with high resolution and reactivity and an accurate data processing system in the scanning mode must also be further improved for the retrospective identification of unknown agents.

Retrospective detection for V-type OPNAs exposure via phosphonylation and disulfide adducts in albumin.

Organophosphorus nerve agents (OPNAs) that damage the central nervous system by inhibiting acetylcholinesterase activity, pose severe threats to human health and life security. Reliable biomarkers that quickly and accurately detect OPNAs exposure are urgently needed to help diagnose quickly and treat in time. Albumins that covalently bind to OPNAs could serve as important targets for retrospective verification of OPNAs exposure. The goal of this study is to explore the potential biomarkers in albumins with high reactivity and good stability and expand the group of potential biomarkers in different species for detecting the exposure of V-type OPNAs including O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). Taking human serum albumin (HSA), bovine serum albumin (BSA) and rabbit serum albumin (RSA) as the research objectives, multiple active sites including phosphonylation and disulfide adduct sites were observed in albumins from different species. Numerous phosphonylation sites labeled by all agents in one type of albumin were found. Among the different species, four shared phosphonylation sites with high reactivity include K499, K549, K249, and Y108. In addition, Y108 on ETY*GEMADCCAK, Y287 on Y*ICENQDSISSK, Y377 on TY*ETTLEK and Y164 on YLY*EIAR in HSA were stably phosphonylated by all agents in gradient concentration, making them stable and suitable potential biomarkers for V-type OPNAs exposure. Notably, Y108 on ETY*GEMADCCAK in HSA, on DTY*GDVADCCEK in RSA, and on ETY*GDMADCCEK in BSA were highly reactive to all V-type agents, regardless of species. It was also successfully labeled in HSA exposed to class V agents in gradient concentration. Y108 is expected to be used to screen and identify the exposure of V-type agents in the retrospective research. Disulfide adducts sites, consisted of four sites in HSA and two sites in BSA were also successfully labeled by V-type agents, and characteristic ion fragments from these disulfide adducts were also identified by secondary mass spectrometry. Molecular simulation of the stably modified sites were conducted to discover the promoting factors of covalent adduct formation, which help further clarify formation mechanism of albumin adducts at active sites.

Simultaneous determination of trace level riot control agents in environmental water by solid-phase microextraction and gas chromatography coupled with a flame ionization detector.

In this paper, a direct immersion solid-phase microextraction procedure for the simultaneous analyses of four primary riot control agents: 2-chloroacetophenone, o-chlorobenzylidene malonitrile, dibenz(b,f)-1,4-oxazepine, and oleoresin capsicum at µg/L concentration from environmental water was developed. Several parameters that influence the extraction effectiveness were investigated, including fiber type, extraction temperature, extraction time, starring rate, and salinity. Under the recommended conditions, the optimized method had reasonable linearity and accuracy. The average recovery of this method ranged from 84 to 108.1%. The limit of detection for all the analytes ranged from 0.2 to 3 µg/L and the limit of quantification ranged from 1 to 10 µg/L, respectively. A relative standard deviation from 3.0 to 4.3% can be achieved depending on the compounds. The procedure was applied to analyze all the four riot control agents simultaneously in several environmental samples.

Forensic signatures of a chemical weapon precursor DMPADC for determination of a synthetic route.

Chemical forensics has been widely recognized as an important tool to investigate alleged use of chemical weapons and/or to identify the illicit production of chemical warfare agents. This paper describes the use of gas chromatography and mass spectrometry (GC-MS) to determine chemical attribution signatures (CAS) N,N-dimethylphosphoramidic dichloride (DMPADC), a key precursor of tabun, for tracking the production of tabun. Synthetic samples were identified and classified by using GC-MS and chemometrics. Analysis samples (n = 27) were collected from three synthetic DMPADC routes; 20 potential CAS were identified, and the structures of five CAS were assigned. Principal component analysis (PCA) was performed to summarize the distribution trend of the samples and to check for the presence of outliers. A Partial least squares discriminant analysis (PLSDA) model was established to discriminate and classify the synthetic samples. The proposed model in this paper has high predictive ability, and the test set samples can be correctly categorized.

Sensitive Untargeted Screening of Nerve Agents and Their Degradation Products Using Liquid Chromatography-High Resolution Mass Spectrometry.

Nerve agents (NAs) are notorious chemical warfare agents that pose a serious threat to national security and public health. The total number of theoretical chemicals of NAs and their degradation products (DPs) exceeds 410 000, according to 1.A.01-1.A.03 in the Schedules of Chemicals of the Chemical Weapons Convention, which poses great challenges for identification and verification. A three-step integrated untargeted screening strategy was developed based on high-resolution mass spectrometry. First, an extensible homemade library for targeted screening of common classical agents was established. Second, a set of in-source collision-induced dissociation mass spectrometry (MS)-alerting ions was extracted and concluded based on fragmentation behavior studies, which included 40 specific alerting ions and 10 types of characteristic structural fragments from total NAs and their DPs. A novel "alerting ion" searching method was developed to rapidly and sensitively screen whether or not nerve agent-related compounds were present and of which type they were. Third, we built a theoretical exact mass database including 202 accurate masses or molecular formulas, which could cover all structural possibilities of the NAs and their DPs. Comprehensively, the elemental composition of pseudomolecular ions, fragment ions, MS/MS spectra, and isotope pattern information were obtained from the full scan MS/data dependent-MS2 experiments and elucidated for identification of the candidates selected in the screening step. This strategy was successfully applied to the identification of unknown chemicals in real samples with good stability and a low limit of detection of 1-10 ng/mL. These procedures are applicable for trace forensic investigations in cases of the alleged use of nerve agents.

Identification of S419 on human serum albumin as a novel biomarker for sarin and cyclosarin exposure.

RATIONALE: Organophosphorus nerve agents are highly toxic because they inhibit acetylcholinesterase activity, thereby causing a series of symptomatic poisoning. Upon entering the body, nerve agents bind active amino acid residues to form phosphonylated adducts. A potentially beneficial method for specific verification of exposure of nerve agents is based on albumin adducts, which have a half-life of 18 days. This appears to be more effective than the fluoride reactivation method, based on acetylcholinesterase. METHODS: After the exposure of human serum albumin to nine nerve agents, human serum albumin was denatured, reduced, alkylated and digested with trypsin according to standard mass spectrometry-based proteomics procedures. The phosphonylated peptides of human serum albumin were identified using positive ion electrospray ionization with a quadrupole orbitrap mass spectrometer. RESULTS: The peptide KVPQVSTPTLVESR showed a good mass spectrometric response to the nine nerve agents. The tendency of sarin and cyclosarin was to bind to S419 on the peptide, while the other nerve agents (tabun, soman and V-type nerve agents) were shown to bind more readily to K414 on the peptide. CONCLUSIONS: This research revealed a new site, S419, of the tryptic peptide KVPQVSTPTLVEVSR on human albumin to be a valuable biomarker for sarin/cyclosarin exposure, helping to further distinguish sarin and cyclosarin poisoning from that of other nerve agents and providing an important tool for the identification of sarin or cyclosarin in terrorist attacks.

Protein adduct binding properties of tabun-subtype nerve agents after exposure in vitro and in vivo.

Upon entering the body, nerve agents can bind active amino acid residues to form phosphonylated adducts. Tabun derivatives (O-alkyl-N,N-dialkyl phosphoroamidocyanidates) have strikingly different structural features from other G-series nerve agents, such as sarin and soman. Here, we investigate the binding mechanism for the phosphonylated adducts of nerve agents of tabun derivatives. Binding sites for three tabun derivatives, O-ethyl-N,N- dimethyl phosphoramidocyanidate (GA), O-ethyl-N,N-ethyl(methyl) phosphoramidocyanidate, and O-ethyl-N,N-diethylphosphoramidocyanidate were studied. Quadrupole-orbitrap mass spectrometry (Q-Orbitrap-MS) coupled to proteomics was used to screen adducts between tabun derivatives and albumin, immunoglobulin, and hemoglobin. The results reveal that all three tabun derivatives exhibit robust selectivity to lysine residues, rather than other amino acid residue types. A set of 10 lysine residues on human serum albumin are labeled by tabun derivatives in vitro, with K525 (K*QTALVELVK) and K199 (LK*CASLQK) peptides displaying the most reactivity. Tabun derivatives formed stable adducts on K525 and K414 (K*VPQVSTPTLVEVSR) for at least 7 days and on K351 (LAK*TYETTLEK) for at least 5 days in a rabbit model. Three of these peptides-K525, K414, and K351-have the highest homology with human serum albumin of all 5 lysine residues that bound to examined rabbit blood proteins in vivo. Molecular simulation of the tabun-albumin interaction using structural analysis and molecular docking provided theoretical evidence supporting lysine residue reactivity to phosphonylation by tabun derivatives. K525 has the lowest free binding energy and the strongest hydrogen bonding to human albumin. In summary, these findings identify unique binding properties for tabun derivatives to blood proteins.

Tracing and attribution of V-type nerve agents in human exposure by strategy of assessing the phosphonylated and disulfide adducts on ceruloplasmin.

V-type agents are highly toxic organophosphorus nerve agents that inhibit acetylcholinesterase in the nervous system, causing a series of poison symptoms. Trace analytical methods are essential for the specific verification of exposure to these agents, especially for human exposure. This paper investigates the phosphonylated and disulfide adducts between human ceruloplasmin and O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2-(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). After being digested by trypsin, the mixture of peptides was separated by a nano-liquid chromatography (nano-LC) and analyzed using quadrupole-orbitrap mass spectrometry (Q-Orbitrap-MS). The sensitive LC-MS/MS-assisted proteomics approach was developed to achieve the identification of human exposure to V-type agents based on these modified sites; results revealed that potential biomarkers could be derived from adducts based on the sulfur- and phosphorus-containing groups of V-type agents. This work offered a novel insight into the mechanism of disulfide-containing adducts resulting from the replacement of disulfide bridges by the thiolate groups from the V-type agents. Moreover, four disulfide adducts on human ceruloplasmin were also discovered during this research, specifically confirming exposure to the V-type agents. Furthermore, molecular simulation testified to the reactivity of the modified sites. Collectively, our findings suggest that the eleven binding sites on human ceruloplasmin have the potential use as a selective marker for prediction the V-type agent exposure in humans.

Verification of soman-related nerve agents via detection of phosphonylated adducts from rabbit albumin in vitro and in vivo.

A major challenge in organophosphate compound (OP) and OP nerve agent (OPNA) research has been in the identification and utilization of reliable biomarkers for rapid, sensitive, and efficient detection of OP exposure. Albumin has been widely studied as a biomarker for retrospective verification of exposure to OPNAs, including soman (GD), by detecting the phosphonylation of specific amino acid residues. The aim of the present study was to identify binding sites between GD and rabbit serum albumin in vitro and in vivo. A nano-liquid chromatography coupled with a quadrupole-orbitrap mass spectrometry (nLC-Q-Orbitrap-MS) was used to examine the GD-modified adducts of rabbit albumin. A total of 11 GD-modified sites were found in rabbit serum albumin across three experimental models. The following five GD-modified rabbit albumin sites, which were all lysine residues, were established in vivo: K188, K329, K162, K233, and K525. Two of these five lysine residues, K188 in peptide EK*ALISAAQER and K162 in peptide YK*AILTECCEAADK, were stable for at least 7 days in vivo. Molecular simulation of the GD-albumin interaction provided theoretical evidence for reactivity of the identified lysine residues. The findings suggest that these modifiable lysine residues are potential biomarkers of GD exposure for retrospective analysis by Q-Orbitrap-MS.

A Novel Potential Biomarker on Y263 Site in Human Serum Albumin Poisoned by Six Nerve Agents.

Albumin is a new biomarker of organophosphorus compounds (OPs) and nerve agents (OPNAs) for retrospective verification. Recent studies on OPs adducts show that amino acid residues can covalently bind to OPs and OPNAs. In this article, after being incubated with soman, sarin, cyclosarin, VX, ethyl tabun, and propyl tabun, human serum albumin (HSA) is analyzed by quadrupole-Orbitrap mass spectrometer (Q Exactive LC-MS/MS). In addition to the three known phosphonylated sites, six new sites modified by OPNAs are detected. To identify the most reactive residue, we calculate the area ratio of the modified peptides to the whole peptides. The result demonstrates that tyrosine 263 (Y263) in peptide Y263ICENQDSISSK, which has been poisoned with six kinds of nerve agents, possesses the highest reactivity. The structure characteristics based on molecular simulation provide a theoretical evidence for the reactivity of the nine binding sites. It suggests that Y263 also has the potential to be used as a biomarker to detect OPNAs exposure, and the presented Q Exactive LC-MS/MS method might be of relevance for the verification of new phosphonylated sites.

α,ß-Unsaturated N-Acylindoles: An Alternative Class of Michael Acceptors and Their Application in Asymmetric Borylation.

Copper(I)-catalyzed enantioselective borylation of α,ß-unsaturated N-acylindoles as well as N-acylpyrroles was efficiently achieved by means of bis(pinacolato)diboron (B2pin2), affording the enantioenriched products in excellent yields with up to 99% ee. The present work provides an alternative class of Michael acceptors, that is, α,ß-unsaturated N-acylindoles, for potential asymmetric transformations.

Mass spectral characterization of tabun-labeled lysine biomarkers in albumin.

Tabun has been shown to form phosphylated adducts on tyrosine residues in albumin in vivo and in vitro. However, in this work, tabun-labeled lysine adducts were found in albumin. Three types of albumin were treated with overdose of tabun in vitro and 17 tabun-labeled lysine residues were found: K4, K12, K224, K377, and K524 in bovine albumin, K186, K188, K212, K329, K414, and K525 in leporine albumin, and K79, K186, K188, K212, K376, and K525 in rat albumin. To investigate the modification of tabun in vivo, three leporines were injected with 0.8×LD50 dose of tabun. The results showed that the labeled lysine residues in vivo, were consistent with modified lysines in vitro. Structure characteristics and the binding mode of 6 tabun-labeled lysines of leporine albumin were further analyzed using theory simulation and molecular docking in Discovery Studio. For the first time, we show that tabun-labeled lysine peptides are found in vivo and in vitro. These modified lysine peptides are good biomarkers for exposure to tabun in albumin of leporine and rat.

Identification of new binding sites of human transferrin incubated with organophosphorus agents via Q Exactive LC-MS/MS.

Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC-MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC-MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC-MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs.

Kinetic and products study of the gas-phase reaction of Lewisite with ozone under atmospheric conditions.

The rate constant for the gas-phase reaction of O3 and Lewisite was studied in air using the smog chamber technique. The experiments were carried out under pseudo-first-order reaction conditions with [O3]≪[Lewisite]. The observed rate constant of O3 with Lewisite was (7.83 ± 0.38) × 10(-19)cm(3)/(molecule·sec) at 298 ± 2K. Lewisite was discussed in terms of reactivity with O3 and its relationship with the ionization potential. Our results show that the rate constant for the gas-phase reaction of O3 with Lewisite is in line with the trend of the rate constants of O3 with haloalkenes.