Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment.

Reductive amination of ω-conotoxin MVIIA: synthesis, determination of modification sites, and self-assembly.

Peptide drugs have disadvantages such as low stability, short half-life and side effects, which limit their widespread use in clinical practice. Therefore, peptide drugs can be modified to improve these disadvantages. Numerous studies have shown that alkyl-modified peptide drugs can self-assemble to prolong the duration of efficacy and/or reduce side effects. However, the commonly used solid-phase synthesis method for alkyl-modified peptides is time-consuming. To overcome this, a simple reductive amination reaction was employed, which can directly graft the alkyl chain to the peptide sequence and effectively avoid stepwise synthesis from C- to N-terminal with amino acids. In this study, ω-conotoxin MVIIA was used as the peptide drug, while myristic aldehyde was used as the alkylating agent. To obtain the maximum productivity of modified peptides, the molar ratio of peptide MVIIA to myristic aldehyde in the reductive amination reaction was optimized. Furthermore, the peptide modification sites in this reaction were confirmed by secondary mass spectrometry analysis. Besides, alkyl-modified peptide MVIIA was able to form micelles by self-assembly and improved stability in serum, which was related to our previous work where myristoylated peptide MVIIA micelles can improve the drug stability. Finally, this study was intended to provide a methodological basis for modifying the alkyl chain of peptide drugs.

Differential Proteomic Analysis of Low-Dose Chronic Paralytic Shellfish Poisoning.

Shellfish poisoning is a common food poisoning. To comprehensively characterize proteome changes in the whole brain due to shellfish poisoning, Tandem mass tag (TMT)-based differential proteomic analysis was performed with a low-dose chronic shellfish poisoning model in mice. A total of 6798 proteins were confidently identified, among which 123 proteins showed significant changes (fold changes of >1.2 or <0.83, p < 0.05). In positive regulation of synaptic transmission, proteins assigned to a presynaptic membrane (e.g., Grik2) and synaptic transmission (e.g., Fmr1) changed. In addition, altered proteins in nervous system development were observed, suggesting that mice suffered nerve damage due to the nervous system being activated. Ion transport in model mice was demonstrated by a decrease in key enzymes (e.g., Kcnj11) in voltage-gated ion channel activity and solute carrier family (e.g., Slc38a3). Meanwhile, alterations in transferase activity proteins were observed. In conclusion, these modifications observed in brain proteins between the model and control mice provide valuable insights into understanding the functional mechanisms underlying shellfish poisoning.

Discriminative detection of soman or VX exposure using europium chelated microparticle-based immunofluorescence microfluidic chip.

The retrospective detection of organophosphorus nerve agents (OPNAs) exposure has been achieved by the off-site analysis of OPNA-human serum albumin (HSA) adducts using mass spectrometry-based detection approaches. However, few specific methods are accessible for on-site detection. To address this, a novel immunofluorescence microfluidic chip (IFMC) testing system combining europium chelated microparticle (EuCM) with self-driven microfluidic chip assay has been established to unambiguously determine soman (GD) and VX exposure within 20 min, respectively. The detection system was based on the principle of indirect competitive enzyme-linked immunosorbent assay. The specific monoclonal antibodies that respectively recognized the phosphonylated tyrosine 411 of GD-HSA and VX-HSA adducts were labeled by EuCM to capture corresponding adducts in the exposed samples. The phosphonylated peptides in the test line and goat-anti-rabbit antibody in the control line were utilized to bind the EuCM-labeled antibodies for signal exhibition. The developed IFMC chip could discriminatively detect exposed HSA adducts with high specificity, demonstrating a low limit of detection at exposure concentrations of 0.5 × 10-6 mol/L VX and 1.0 × 10-6 mol/L GD. The exposed serum samples can be qualitatively detected following an additional pretreatment procedure. This is a novel rapid detection system capable of discriminating GD and VX exposure, providing an alternative method for rapidly identifying OPNA exposure.

A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.

Study on the Analgesic Activity of Peptide from Conus achates.

BACKGROUND: As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons. OBJECTIVE: To recognize the target protein and analgesic activity of Ac6.4 from Conus achates. METHODS: In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice. RESULTS: Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 µg/kg) and Trx-Ac6.4 (20, 40, 80 µg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent. CONCLUSION: This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs.

Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL-1, 0.28 ng mL-1, 0.50 ng mL-1, 0.41 ng mL-1 and 0.91 ng mL-1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00-400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Simultaneous quantification of multiple amino acid adducts from sulfur mustard-modified human serum albumin in plasma at trace exposure levels by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry after propionyl derivatization.

Sulfur mustard (HD) is a highly toxic vesicant and is prohibited by the Organisation for the Prohibition of Chemical Weapons (OPCW). HD can modify human serum albumin (HSA) to generate hydroxyethylthioethyl (HETE) adducts, which could be utilized as biomarkers for verifying HD exposure in forensic analysis. Here, five amino acid adducts generated from pronase digestion of HD-exposed human serum albumin (HD-HSA) in plasma were selected as biomarkers to retrospectively detect HD exposure. HD-HSA was precipitated from plasma with acetone, digested by pronase, derivatized with propionic anhydride (PA), and analysed with ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-TQ MS). The limits of detection (LODs) and limits of quantification (LOQs) of the HD exposure concentrations were evaluated as 1.00 ng/mL at S/N≥3 and 3.00 ng/mL at S/N≥10, respectively, which are approximately 60 times lower than those of the reported method. The approach shows good linearity (R2≥0.997) from 3.00 ng/mL to 10.0 µg/mL of HD-exposed human plasma with satisfactory precision and accuracy. The developed approach was applied to analysing samples from the 6th OPCW Biomedical Proficiency Test (BioPT). The study showed that the developed approach was also suitable for analysing human plasma samples that were exposed to six of HD analogues, which were common impurities in sulfur mustard mixtures. Moreover, the method was successfully applied to plasma from other species, including rabbits, rats and cattle. This study provides a reliable and sensitive tool for the retrospective detection of vesicants exposure based on multiple biomarkers.

A proteomics strategy for the identification of multiple sites in sulfur mustard-modified HSA and screening potential biomarkers for retrospective analysis of exposed human plasma.

A major challenge for the unequivocal verification of alleged exposure to sulfur mustard (HD) lies in identifying its multiple modifications on endogenous proteins and utilizing these modified proteins to achieve accurate, sensitive, and rapid detection for retrospective analysis of HD exposure. As the most abundant protein in human plasma, human serum albumin (HSA) can react with many xenobiotics, such as HD, to protect the body from damage. The HSA adducts induced by HD have been used as biomarkers for the verification of HD exposure. In this study, the modification sites on HSA by HD were identified through application of the bottom-up strategy used in proteomics, and 41 modified sites were discovered with seven types of amino acids, of which 3 types were not previously reported. Then, different enzymes, including pepsin, endoproteinase Glu-C, and pronase, were applied to digest HD-HSA to produce adducts with hydroxyethylthioethyl (HETE) groups, which may be used as potential biomarkers for HD exposure. As candidates for retrospective analysis, sixteen adducts were obtained and characterized with ultra-high-pressure liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (UHPLC-QE Focus MS). These potential biomarkers were evaluated in human plasma that was exposed in vitro to HD and five of its analogues. This study integrated the identification of modification sites through application of the bottom-up strategy of proteomics and screening biomarkers, providing a novel strategy for retrospective detection of the exposure of xenobiotic chemicals.

Simultaneous solvent extraction and quantification of eleven amine compounds related to Chemical Weapon Convention in soils via hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The detection of Chemical Weapon Convention (CWC)-related amine compounds including the precursors or degradation products of V-type organophosphorus nerve agent, nitrogen mustard and 3-quinuclidinyl benzilate is an important aspect for verifying their intact chemical warfare agents. This work focuses on the development of a novel formulation for the simultaneous solvent extraction of eleven CWC-related amine compounds, from the four-type soil matrices including environmental standard soil, sand, clay, and loam. Extracts were well separated on the hydrophilic interaction liquid chromatography (HILIC) and then detected by MS/MS multiple reaction monitoring mode. The type and component of solvent mixtures were optimized to cover a wide range of polarity over all eleven amine compounds with high extraction efficiencies. Extraction parameters, such as the proportion of methanol, water and NH4OH, the times and the period of extraction, and volumes of extraction solution were optimized. The results indicated that a mixed solvent of methanol/water (44:53, v/v) in 3.0% NH4OH was the optimal formulation for extraction of all 11 analytes with high mean extraction recoveries (64.4-96.1%). Specificity and sensitivity were well improved by the good separation of 11 analytes from four-type soil matrices using these optimized HILIC parameters. This method was fully validated for each analyte in four soil matrices. The linear range of 11 analytes was 0.50/0.75-500 ng·g-1 with correlation coefficient (R2) ≥0.990, and intra/inter-day accuracies were 70.3-125% with relative standard deviation (RSD) ≤19.3%. Limit of detection (LOD) of 11 analytes ranged from 0.01 to 0.5 ng·g-1, which was far lower than those reported in previous studies. The built method accomplishes simultaneously quantitative and trace measurement of all eleven CWC-related amine compounds within a single solvent extraction and detection. It only takes a small amount of soil samples and possesses the highest sensitivity over all previous methods. This study provides an optional recommended operating procedure for determination of CWC-related amine compounds in four typical types of complex soils during chemical weapons verification.

Screening of monoclonal antibodies against specific phosphonylation sites and analysis of serum samples exposed to soman and VX using an indirect competitive enzyme-linked immunosorbent assay.

Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6 mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6 mol/L and 10.0 × 10-6 mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.

[Highly toxic type â ¡ ribosome-inactivating proteins ricin and abrin and their detection methods: a review].

Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has N-glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity. Ricin and abrin that are expressed by the plants Ricinus communis and Abrus precatorius, respectively, are typical type Ⅱ RIPs. The toxicity of ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years. Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on N-glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis.

The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1-1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

Advances in sulfur mustard-induced DNA adducts: Characterization and detection.

Sulfur mustard (SM) is a blister chemical warfare agent with severe cytotoxicity and genotoxicity. It can extensively alkylate important macromolecules in organisms, such as proteins, DNA, and lipids, and produce a series of metabolites, among which the characteristic ones can be used as biomarkers. The exact toxicological mechanisms of SM remain unclear but mainly involve the DNA lesions induced by alkylation and oxidative stress caused by glutathione depletion. Various methods have been used to analyze DNA damage caused by SM. Among these methods, liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology stands out and makes it possible to observe damage in view of biomarkers induced by SM. Sample preparation is critical for detection by LC-MS/MS and mainly includes DNA isolation, adduct hydrolysis, and adduct purification. Moreover, optimization of chromatographic conditions, selection of MS transitions, and quantitative strategies are also essential. SM-DNA adducts are generally considered to be N7-HETEG, O6-HETEG, N7-BisG, and N3-HETEA. This article proposes some other possibilities of SM-DNA adducts for the identification of SM genotoxicity.

Quantitative detection of ricin in beverages using trypsin/Glu-C tandem digestion coupled with ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS. The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion. Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1 ng/mL in PBS and 0.5 ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5-300 ng/mL in PBS, 1.0-400 ng/mL in milk, and 1.0-250 ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9-105.2% for milk, and 95.3-118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3-9.4%, 0.7-8.9%, and 0.2-6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.

Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC-MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin.

Ricin is a type II ribosome-inactivating protein toxin consisting of A and B chains linked by one interchain disulfide bond. Because of its high toxicity depending on both chains together, confirming the presence of both A and B chains of intact ricin is required during the investigation of the illegal production and application. Here, we report a novel and sensitive acetonitrile (ACN)-assisted trypsin digestion method for unambiguous identification of intact ricin by simultaneous detection of its marker peptides from A and B chains. Marker peptides were generated with a simple procedure by direct cleaving the native ricin at 45 °C for 4 h using Promega modified sequencing grade trypsin under the assistance of 10% ACN, and then directly analyzed by ultrahigh performance liquid chromatography tandem mass spectrometry. The type of trypsin was found to be one critical factor for cleavage of intact ricin based on a significant difference in the yields of specific peptides generated while using various types of trypsin. A low content of ACN in enzymatic buffer significantly reduced the digestion time from overnight to 4 h. There was commonly a better MS response of marker peptides when using the developed ACN-assisted trypsin digestion method than methanol-assisted trypsin digestion within the same 4 h. Totally, seven specific peptides with high sensitivity and specificity including three in the A-chain (TA7, TA11, and TA10) and four in the B-chain (TB6, TB14-ss-TB16, TB20, and TB18) were obtained as good marker peptides for unambiguous identification of intact ricin. The lowest concentration of native ricin for unambiguous identification was 20 ng/mL, in which three marker peptides from both the A-chain and B-chain could be measured with a minimum of three ion transitions. Combined with affinity enrichment, the developed approach was successfully applied for the measurement of intact ricin from the complicated matrix samples of the second, third, and fourth biotoxin exercises organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). This study has provided a recommended detection method combined with one novel ACN-assisted trypsin digestion with MS for forensic unambiguous confirmation of trace ricin intact with high confidence.

Fluoride reactivation-enabled sensitive quantification of tabun adducts on human serum albumin by GC-MS/MS via isotope dilution.

Organophosphorus nerve agents inhibit the cholinesterase activity by phosphylation of the active site serine. The resulting phosphylated cholinesterase and adducts on human serum albumin (HSA) are appropriate biomarkers for nerve agents exposure. Several methods have been developed for the detection of nerve agents, including fluoride reactivation or alkaline cleavage. It was previously thought that some nerve agents adducts to HSA could not be detected via fluoride regeneration. In our study, the results showed that tabun (GA) adducts of HSA could be detected by fluoride regeneration. The sample preparation included acetone precipitation, washing and SPE. Deuterated tabun (d5-GA) was applied as the internal standard. The product of regenerated fluorotabun is detected with a good linearity (R2 > 0.997) in the concentration range from 0.02 to 100.0 ng/ml, small relative standard deviation (≤6.89%) and favorable recoveries between 94.8 and 106.3%. The established preparation confirmed the fluorotabun was regenerated from the GA-HSA adducts.

LC-HRMS Screening and Identification of Novel Peptide Markers of Ricin Based on Multiple Protease Digestion Strategies.

Both ricin and R. communisagglutinin (RCA120), belonging to the type II ribosome-inactivating proteins (RIPs-Ⅱ), are derived from the seeds of the castor bean plant. They share very similar amino acid sequences, but ricin is much more toxic than RCA120. It is urgently necessary to distinguish ricin and RCA120 in response to public safety. Currently, mass spectrometric assays are well established for unambiguous identification of ricin by accurate analysis of differentiated amino acid residues after trypsin digestion. However, diagnostic peptides are relatively limited for unambiguous identification of trace ricin, especially in complex matrices. Here, we demonstrate a digestion strategy of multiple proteinases to produce novel peptide markers for unambiguous identification of ricin. Liquid chromatography-high resolution MS (LC-HRMS) was used to verify the resulting peptides, among which only the peptides with uniqueness and good MS response were selected as peptide markers. Seven novel peptide markers were obtained from tandem digestion of trypsin and endoproteinase Glu-C in PBS buffer. From the chymotrypsin digestion under reduction and non-reduction conditions, eight and seven novel peptides were selected respectively. Using pepsin under pH 1~2 and proteinase K digestion, six and five peptides were selected as novel peptide markers. In conclusion, the obtained novel peptides from the established digestion methods can be recommended for the unambiguous identification of ricin during the investigation of illegal use of the toxin.

A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Sulfur mustard (HD) reacts with human serum albumin (HSA) at Cys34 and produces a long-term biomarker of HD exposure. Here, we present a novel, sensitive, and convenient method for quantification of HD exposure by detection of HD-HSA adducts using pronase digestion, benzyl chloroformate (Cbz-Cl) derivatization, and ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The HSA in HD-exposed plasma in vitro was precipitated with acetone and digested (2 h, 50 °C) with pronase to form the alkylated dipeptide, S-hydroxyethylthioethyl-CysPro (HETE-CP). The HETE-CP adduct was derivatized with Cbz-Cl to generate N-carbobenzoxy HETE-CP (HETE-C(Cbz)P). The derivatized product was analyzed by UHPLC-MS/MS. HD surrogate, 2-chloroethyl ethyl sulfide (2-CEES), was introduced as a non-isotope internal standard (ISTD) instead of traditional d8-HD for quantification. The method was found to be linear between 1.00 and 200 ng/mL HD exposure (R2 > 0.998) with precision of ≤ 9.0% relative standard deviation (RSD) and accuracy ranged between 97.1 and 111%. The limit of detection (LOD) is 0.500 ng/mL (S/N~5), over 15 times lower than that of the previous method (7.95 ng/mL). Time-consuming affinity purification or solid phase extraction (SPE) is not needed in the experiment and the operation takes less than 5 h. This study provides a new strategy and useful tool for retrospective analysis of HD exposure by HETE-CP biomarker detection. Graphical abstract Flow diagram for quantification of sulfur mustard exposure by detection of HETE-CP dipeptide adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Effect of Temperature, Pressure, and Chemical Composition on the Electrical Conductivity of Schist: Implications for Electrical Structures under the Tibetan Plateau.

The experimental study on the electrical conductivities of schists with various contents of alkali ions (CA = K2O + Na2O = 3.94, 5.17, and 5.78 wt.%) were performed at high temperatures (623⁻1073 K) and high pressures (0.5⁻2.5 GPa). Experimental results indicated that the conductivities of schist markedly increased with the rise of temperature. Pressure influence on the conductivities of schist was extremely weak at the entire range of experimental temperatures. Alkali ion content has a significant influence on the conductivities of the schist samples in a lower temperature range (623⁻773 K), and the influence gradually decreases with increasing temperature in a higher temperature range (823⁻1073 K). In addition, the activation enthalpies for the conductivities of three schist samples were fitted as being 44.16⁻61.44 kJ/mol. Based on the activation enthalpies and previous studies, impurity alkaline ions (K⁺ and Na⁺) were proposed as the charge carriers of schist. Furthermore, electrical conductivities of schist (10-3.5⁻10-1.5 S/m) were lower than those of high-conductivity layers under the Tibetan Plateau (10-1⁻10° S/m). It was implied that the presence of schist cannot cause the high-conductivity anomalies in the middle to lower crust beneath the Tibetan Plateau.