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.

First Display of Haloalkane Dehalogenase LinB on the Surface of Bacillus subtilis Spore.

BACKGROUND: LinB, as a Haloalkane dehalogenase, has good catalytic activity for many highly toxic and recalcitrant compounds, and can realize the elimination of chemical weapons HD in a green non-toxic mode. OBJECTIVES: In order to display Haloalkane dehalogenase LinB on the surface of Bacillus subtilis spore. METHODS: We have constituted the B. subtilis spore surface display system of halogenated alkanes dehalogenase LinB by gene recombination. RESULTS: Data revealed that LinB can display on spore surface successfully. The hydrolyzing HD analogue 2-chloroethyl ethylsulfide (2-CEES) activity of displayed LinB spores was 4.30±0.09 U/mL, and its specific activity was 0.78±0.03U/mg. Meanwhile, LinB spores showed a stronger stress resistance activity on 2-CEES than free LinB. This study obtained B. subtilis spores of LinB (phingobium japonicum UT26) with enzyme activity that was not reported before. CONCLUSION: Spore surface display technology uses resistance spore as the carrier to guarantee LinB activity, enhances its stability, and reduces the production cost, thus expanding the range of its application.

Self-Assembly Nanostructure of Myristoylated ω-Conotoxin MVIIA Increases the Duration of Efficacy and Reduces Side Effects.

Chronic pain is one of the most prevalent health problems worldwide. An alternative to suppress or alleviate chronic pain is the use of peptide drugs that block N-type Ca2+ channels (Cav2.2), such as ω-conotoxin MVIIA. Nevertheless, the narrow therapeutic window, severe neurological side effects and low stability associated with peptide MVIIA have restricted its widespread use. Fortunately, self-assembly endows the peptide with high stability and multiple functions, which can effectively control its release to prolong its duration of action. Inspired by this, MVIIA was modified with appropriate fatty acid chains to render it amphiphilic and easier to self-assemble. In this paper, an N-terminal myristoylated MVIIA (Myr-MVIIA, medium carbon chain length) was designed and prepared to undergo self-assembly. The present results indicated that Myr-MVIIA can self-assemble into micelles. Self-assembled micelles formed by Myr-MVIIA at higher concentrations than MVIIA can prolong the duration of the analgesic effect and significantly reduce or even eliminate the side effects of tremor and coordinated motor dysfunction in mice.

Qualitative Analysis of Novel Flavonoid Adducts from Nerve Agent Tabun-Exposed Arabidopsis thaliana (L.) Based on Quadrupole-Time of Flight Mass Spectrometry.

Flavonoids are a kind of secondary metabolite which widely exist in plants. They contain a lot of active hydroxyls, which can react with toxic chemicals to produce potential exposure biomarkers. In this article, the model plant Arabidopsis thaliana (L.) was exposed to the nerve agent O-Ethyl N,N-dimethyl phosphoramidocyanidate (Tabun). By comparing with the plant not exposed to Tabun, some characteristic ions were identified by quadrupole-time of flight mass spectrometry in the acetonitrile extract of the exposed leaves. These characteristic ions were selected as parent ions to produce product ion mass spectra (PIMS). Some interesting fragmentation pathways were revealed, including neutral loss of glucoside, rhamnose and ethylene. O-Ethyl N,N-dimethyl phosphoryl modified flavonoids were deduced from assignment of the PIMS. The element components and the accurate mass of the product ions from each parent ion matched well with those of the proposed fragmentation pathways. Through comparison with the PIMS of structurally closely related chemical of Isobutyl methylphosphonyl modified flavonoids, the structures and the fragmentation pathways of the O-Ethyl N,N-dimethyl phosphoryl modified flavonoids were finally confirmed. Successfully finding and identifying these three specific exposure biomarkers in plants provided a new strategy for the retrospective analysis of organophosphorus exposure and forensic analysis.

Identification of four novel flavonoid adducts in Arabidopsis thaliana (L.) exposed to isobutyl S-2-diethylaminoethyl methylphosphonothiolate as potential plant exposure biomarkers.

As vegetation is part of our lives, plants are good candidates as indicators of toxic chemicals. Numerous components in plants may react with toxic chemicals to produce exposure biomarkers. Plant biomarkers formed by the modification of endogenous plant components by chemical warfare agents have not been reported. In this article, the model plant Arabidopsis thaliana (L.) was exposed to the nerve agent isobutyl S-2-diethylaminoethyl methylphosphonothiolate (iBuVX). Some characteristic ions were identified by liquid chromatography-high resolution mass spectrometry and their product ion mass spectra were recorded and interpreted. Some interesting fragmentation pathways were revealed including neutral loss of glucoside, rhamnose and isobutylene. Isobutyl methylphosphonyl modified flavonoids were deduced from assignment of product ions. The element components and the accurate mass of the product ions matched well with those of the proposed fragmentation pathways. The binding site of the nerve agent on flavonoids was proved to be the hydroxyl group on the benzene ring of the flavonoids by density functional theory computation and by the synthesis of the reference chemical, which was confirmed by 1H-31P HMBC NMR. The phosphonyl-modified flavonoids were evaluated for specificity in different plants. Four new flavonoid adducts as potential biomarkers were identified in the leaves of the iBuVX-exposed plant, which provided a novel strategy for the retrospective analysis of organophosphorus exposure for chemical weapon verification and forensic analysis.

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.

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.

Facile synthesis and nematicidal activity evaluation of thiophosphinyl amide [(Pz)2P(S)NHR] and thiophosphonyl diamide [(Pz)P(S)(NHR)2] (Pz = 1,3,5-trimethylpyrazole, R = biphenyl derivatives).

A series of thiophosphinyl amide [(Pz)2P(S)NHR] and thiophosphonyl diamide [PzP(S)(NHR)2] compounds, where Pz = 1,3,5-trimethylpyrazole and N(H)R = derivatives of 2-aminobiphenyl, were synthesized via a facile two-step process. Reaction of pyrazolyl substituted bromophosphine with 2-aminobiphenyl derivatives and further reaction with elemental sulphur affords the corresponding thiophosphinyl amide and thiophosphonyl diamide. The intermediate species was used without prior purification for reaction with sulphur to yield the target compounds. The nematicidal activity evaluation suggests that some compounds could manifest moderate nematicidal activity towards Meloidogyne incogita, which is higher than that of their amide analogue bixafen.

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.

Regioselective O/C phosphorylation of α-chloroketones: a general method for the synthesis of enol phosphates and ß-ketophosphonates via Perkow/Arbuzov reaction.

A regioselective O/C phosphorylation of α-chloroketones with trialkyl phosphites was performed for the first time, which employed solvent-free Perkow reaction and NaI-assisted Arbuzov reaction under mild conditions respectively. Versatile enol phosphates were prepared in good to excellent yields as well as ß-ketophosphinates.

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.

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.

Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA.

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.

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.