Disulfide-adducts with cysteine residues in human serum albumin prove exposure to malodorous mercaptans in vitro.

Malodorants are mixtures containing mercaptans, which trigger the flight instinct upon exposure and might thus be deployed in military and civilian defense scenarios. Exposure to mercaptans might lead to unconsciousness, thus representing a possible threat for health. Therefore, we developed and validated a bioanalytical procedure for the simultaneous detection and identification of corresponding biomarkers for the verification of exposure to mercaptans. Disulfide-adducts of ethyl mercaptan (SEt), n-butyl mercaptan (SnBu), tert-butyl mercaptan (StBu) and iso-amyl mercaptan (SiAm) with cysteine (Cys) residues in human serum albumin (HSA) were formed by in vitro incubation of human plasma. After pronase-catalyzed proteolysis, reaction products were identified as adducts of the single amino acid Cys and the dipeptide cysteine-proline (Cys34Pro) detected by a sensitive µLC-ESI MS/MS method working in the scheduled multiple reaction monitoring (sMRM) mode. Dose-response studies showed linearity for the yield of Cys34Pro-adducts in the range from 6 nM to 300 µM of mercaptans in plasma and limits of identification (LOI) were in the range from 60 nM to 6 µM. Cys34-adducts showed stability for at least 6 days in plasma (37 °C). The presented disulfide-biomarkers expand the spectrum for bioanalytical verification procedures and might be helpful to prove exposure to malodorants.

Effects of the nerve agent VX on hiPSC-derived motor neurons.

Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date, the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation, motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity, MN were initially exposed once to 400 µM, 600 µM, 800 µM, or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS, XTT, IncuCyte, qRT-PCR, and Western Blot. For the first time, VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this, MN morphology and neurite network were altered time and concentration-dependently. Thus, MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future.

The phosphylated butyrylcholinesterase-derived tetrapeptide GlyGluSerAla proves exposure to organophosphorus agents with enantioselectivity.

We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer198*Ala generated from butyrylcholinesterase (BChE) with proteinase K excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser198) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8 µm) in micro-liquid chromatography-electrospray ionisation tandem-mass spectrometry (µLC-ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (-)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (-)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.

Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo.

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

Phosphonylated tyrosine and lysine residues as biomarkers of local exposure of human hair to the organophosphorus nerve agents sarin and VX.

We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a µLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9 weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure.

Activation of the human TRPA1 channel by different alkylating sulfur and nitrogen mustards and structurally related chemotherapeutic drugs.

An important target in toxicology is the ion channel known as human transient receptor potential ankyrin 1 (hTRPA1). It is triggered by a variety of chemicals, including the alkylating chemical warfare agent sulfur mustard (SM). The activation potentials of structural analogs including O- and sesquimustard, nitrogen mustards (HN1, HN2, and HN3), and related chemotherapeutic drugs (bendamustine, cycylophosphamide, and ifosfamide) were examined in the current study. The aequorin assay was used to measure changes in intracellular calcium levels in human hTRPA1 overexpressing HEK293 cells. The XTT assay was used to determine cytotoxicity. The data presented here highlight that all investigated alkylating substances, with the exception of cyclophosphamide and ifosfamide, cause the activation of hTRPA1. Cytotoxicity and activation of hTRPA1 were found to be related. Compounds with high reactivity had higher cytotoxicity and vice versa. However, inhibiting hTRPA1 with the specific inhibitor AP18 could not reduce the cytotoxicity induced by alkylating agents. As a result, hTRPA1 does not play a significant role in the cytotoxicity of alkylating agents.

Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro.

Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 °C in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC50) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 µM. In contrast, the exposure to malaoxon in the presence of 1,000 µM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.

Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure.

Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys462 and Cys665, as well as at Asp339 and Glu341 described herein for the first time. Since Cys665 is a well-known target of hTRPA1 agonists and is involved in hTRPA1 activation, SM-induced modifications of cysteine, as well as aspartic acid and glutamic acid residues may play a role in hTRPA1 activation. Considering hTRPA1 as a target of other SM-related chemical warfare agents, analogous adducts may be predicted and identified applying the analytical approach described herein.

Highly stable peptide adducts from hard keratins as biomarkers to verify local sulfur mustard exposure of hair by high-resolution mass spectrometry.

In the recent past, the blister agent sulfur mustard (SM) deployed by the terroristic group Islamic State has caused a huge number of civilian and military casualties in armed conflicts in the Middle East. The vaporized or aerolized agent might be inhaled and have direct contact to skin and hair. Reaction products of SM with plasma proteins (adducts) represent well-established systemic targets for the bioanalytical verification of exposure. The SM-derived hydroxyethylthioethyl (HETE)-moiety is attached to nucleophilic amino acid side chains and allows unambiguous adduct detection. For shipping of common blood and plasma samples, extensive packaging rules are to be followed as these matrices are considered as potentially infectious material. In contrast, hair is considered as non-infectious thus making its handling and transportation much less complicated. Therefore, we addressed this matrix to develop a procedure for bioanalytical verification. Following optimized lysis of SM-treated human scalp hair and pepsin-catalyzed proteolysis of adducts of keratin type I and II, microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) was used to detect three alkylated keratin-derived biomarker peptides: AE(-HETE)IRSDL, FKTIE(-HETE)EL, and LE(-HETE)TKLQF simultaneously. All bear the HETE-moiety bound to a glutamic acid residue. Protein adducts were stable for at least 14 weeks at ambient temperature and contact to air, and were not affected by washing the hair with shampoo. The biomarker peptides were also obtained from beard, armpit, abdominal, and pubic hair. This is the first report introducing stable local peptide adduct biomarkers from hair, that is easily accessible by a non-invasive sampling process.

Alkylated glutamic acid and histidine derived from protein-adducts indicate exposure to sulfur mustard in avian serum.

Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals. For legal reasons, bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins, for example, serum albumin (SA), are valuable long-lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck, and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant, and re-dissolution analysis were performed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry in the selected reaction monitoring mode, µLC-ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE]+ at m/z 105.0. After in vitro incubation with SM and pronase-catalyzed proteolysis, the alkylated amino acids Glu(-HETE) and His(-HETE) were detected. Both borne the SM-characteristic HETE-moiety bound to their side chain. The eightfold deuterated SM analog (d8-SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50°C), and time to maximize the yield of Glu(-HETE) (30 min) and His(-HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein-adducts were stable in serum at -30°C for at least 33 days and for three freeze-and-thaw cycles. At the body temperature of chicken (+40°C), Glu(-HETE) was degraded in serum (period of half-change 3 days), whereas His(-HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM.

Nontargeted High-Resolution Mass Spectrometric Workflow for the Detection of Butyrylcholinesterase-Derived Adducts with Organophosphorus Toxicants and Structural Characterization of Their Phosphyl Moiety after In-Source Fragmentation.

Organophosphorus (OP) nerve agents were used for chemical warfare, assassination, and attempted murder of individuals. Therefore, forensic methods are required to identify known and unknown incorporated OP poisons. Serum is tested for the presence of covalent reaction products (adducts) of the toxicant with, e.g., butyrylcholinesterase (BChE) typically by targeted analysis, thus only detecting known OP adducts. We herein present a nontargeted two-step mass spectrometry (MS)-based workflow taking advantage of a high-resolution (HR) Orbitrap mass spectrometer and its option for in-source collision-induced dissociation (IS-CID) highly valuable for the detection of unknown agents. BChE adducts are extracted by immunomagnetic separation and proteolyzed with pepsin yielding a phosphylated nonapeptide (NP) biomarker NP(OP). In step 1, the sample is separated by micro liquid chromatography (µLC) detecting the NP(OP) by nontargeted HR MS followed by data-dependent tandem-MS (ddMS2). Extracted ion chromatograms of diagnostic product ions at m/z 778.33661, 673.29402, and 602.25690 reveal the accurate mass of the NP(OP) precursor ion as well as the elemental composition of the adducted phosphyl moiety. Considering this information, a second µLC run is performed (step 2) for nonselective IS-CID of NP(OP) yielding the cleaved charged phosphyl moiety. This fragment ion is immediately subjected to targeted CID in parallel reaction monitoring (PRM). The accurate mass of its product ions allows the determination of their elemental composition and thus supports its structural elucidation. The described workflow was exemplarily applied to NP(OP) of three Tamelin esters and VX providing highly appropriate abilities for the detection of adducts even of unknown OP poisons like Novichok agents.

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization.

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 µM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.

A case report of severe pirimiphos-methyl intoxication: Clinical findings and cholinesterase status.

A 63-year-old male was admitted to a district hospital after ingesting ethanol and pirimiphos-methyl (PM) with suicidal intentions. History included alcoholic cirrhosis with alcoholism, adiposity, diabetes with cerebral microangiopathy, chronic renal insufficiency, heparin-induced thrombocytopenia, and status post necrotizing fasciitis. Emergency medical service reported an alert patient without signs of cholinergic crisis; activated charcoal and atropine were administered. Upon hospital arrival, he received fluid resuscitation, activated charcoal, and atropine. He was transferred to a toxicology unit the next day. On admission, he had no cholinergic signs (dry mucous membranes, warm skin, and mydriatic pupils) requiring small atropine doses (0.5 mg per hour). Four hours after admission, he developed bradycardia and respiratory distress, necessitating intubation. He received atropine by continuous infusion for 7 days (248 mg total) and obidoxime (bolus and continuous infusion). PM, pirimiphos-methyl-oxon (PMO), and phosphorylated tyrosine (Tyr) adducts derived from human serum albumin were analyzed in vivo. Cholinesterase status (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), inhibitory activity of patient plasma and reactivatability, and phosphorylated BChE-derived nonapeptides) was measured in vivo. Obidoxime and atropine were monitored. PM and PMO were detectable, PM with maximum concentration ∼24 h post admission (p.a.) and PMO at ∼18 h p.a. Tyr adducts were detectable. AChE in vivo was suppressed on admission, increased continuously after starting obidoxime, and reached maximum activity after ∼30 h. AChE in vivo and reactivatability remained at the same level until the end of monitoring. BChE was already suppressed on admission; termination of the antidote treatment was possible after BChE had recovered to 1/5th of its normal value and extubation was possible after BChE had recovered to 2/5th. While a substantial part of BChE was already aged on admission, aging continued peaking at ∼24 h p.a. After initiating obidoxime treatment, plasma levels increased until obidoxime plasma levels reached a steady state. On admission, plasma atropine level was low; it increased with the start of the continuous infusion. Afterward, the level dropped to a steady state. The clinical course was characterized by bouts of pneumonia, necessitating re-intubation and prolonged ventilation, sepsis, delirium, and a peripheral neuropathy. After psychiatric evaluation, the patient was discharged to a neurological rehabilitation facility after 77 days of hospital care.

Poisoning by organophosphorus nerve agents and pesticides: An overview of the principle strategies and current progress of mass spectrometry-based procedures for verification.

Intoxication by organophosphorus (OP) poisons, like nerve agents and pesticides, is characterized by the life-threatening inhibition of acetylcholinesterase (AChE) caused by covalent reaction with the serine residue of the active site of the enzyme (phosphylation). Similar reactions occur with butyrylcholinesterase (BChE) and serum albumin present in blood as dissolved proteins. For forensic purposes, products (adducts) with the latter proteins are highly valuable long-lived biomarkers of exposure to OP agents that are accessible by diverse mass spectrometric procedures. In addition, the evidence of poison incorporation might also succeed by the detection of remaining traces of the agent itself, but more likely its hydrolysis and/or enzymatic degradation products. These relatively short-lived molecules are distributed in blood and tissue, and excreted via urine. This review presents the mass spectrometry-based methods targeting the different groups of biomarkers in biological samples, which are already internationally accepted by the Organisation for the Prohibition of Chemical Weapons (OPCW), introduces novel approaches in the field of biomedical verification, and outlines the strict quality criteria that must be fulfilled for unambiguous forensic analysis.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro.

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

Alkylated albumin-derived dipeptide C(-HETE)P derivatized by propionic anhydride as a biomarker for the verification of poisoning with sulfur mustard.

Sulfur mustard (SM) is a banned chemical warfare agent recently used in the Syrian Arab Republic conflict causing erythema and blisters characterized by complicated and delayed wound healing. For medical and legal reasons, the proof of exposure to SM is of high toxicological and forensic relevance. SM reacts with endogenous human serum albumin (HSA adducts) alkylating the thiol group of the cysteine residue C34, thus causing the addition of the hydroxyethylthioethyl (HETE) moiety. Following proteolysis with pronase, the biomarker dipeptide C(-HETE)P is produced. To expand the possibilities for verification of exposure, we herein introduce a novel biomarker produced from that alkylated dipeptide by derivatization with propionic anhydride inducing the selective propionylation of the N-terminus yielding PA-C(-HETE)P. Quantitative derivatization is carried out at room temperature in aqueous buffer within 10 s. The biomarker was found to be stable in the autosampler at 15 °C for at least 24 h, thus documenting its suitability even for larger sets of samples. Selective and sensitive detection is done by micro liquid chromatography-electrospray ionization tandem-mass spectrometry (µLC-ESI MS/MS) operating in the selected reaction monitoring (SRM) mode detecting product ions of the single protonated PA-C(-HETE)P (m/z 379.1) at m/z 116.1, m/z 137.0, and m/z 105.0. The lower limit of detection corresponds to 32 nM SM in plasma in vitro and the limit of identification to 160 nM. The applicability to real exposure scenarios was proven by analyzing samples from the Middle East confirming poisoning with SM.

Evidence of sulfur mustard poisoning by detection of the albumin-derived dipeptide biomarker C(-HETE)P after nicotinylation.

Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent that was frequently used in recent years and led to numerous poisoned victims who developed painful erythema and blisters. Post-exposure analysis of SM incorporation can be performed by the detection of human serum albumin (HSA)-derived peptides. HSA alkylated by SM contains a hydroxyethylthioethyl (HETE)-moiety bound to the cysteine residue C34 yielding the dipeptide biomarker C(-HETE)P after pronase-catalyzed proteolysis. We herein present a novel procedure for the selective precolumn nicotinylation of its N-terminus using 1-nicotinoyloxy-succinimide. The reaction was carried out for 2 h at ambient temperature with a yield of 81%. The derivative NA-C(-HETE)P was analyzed by micro liquid chromatography-electrospray ionization tandem-mass spectrometry working in the selected reaction monitoring mode (µLC-ESI MS/MS SRM). The derivative was shown to be stable in the autosampler at 15°C for at least 24 h. The single protonated precursor ion (m/z 428.1) was subjected to collision-induced dissociation yielding product ions at m/z 116.1, m/z 137.0, and m/z 105.0 used for selective monitoring without any plasma-derived interferences. NA-C(-HETE)P showed a mass spectrometric response superior to the non-derivatized dipeptide thus yielding larger peak areas (factor 1.3 ± 0.2). The lower limit of identification corresponded to 80 nM SM spiked to plasma in vitro. The presented procedure was applied to real case plasma samples from 2015 collected in the Middle East confirming SM poisoning.

Evidence of exposure to organophosphorus toxicants by detection of the propionylated butyrylcholinesterase-derived nonapeptide-adduct as a novel biomarker.

Organophosphorus (OP) nerve agents represent a class of highly toxic chemical warfare agents banned by the Chemical Weapons Convention. Nevertheless, in the past few years they have been used repeatedly for warfare, assassination and attempted murder. In addition, the chemically related OP pesticides were frequently used for suicide and may be deployed for terroristic attacks. Therefore, sensitive and selective bioanalytical methods are indispensable to investigate biological specimens as pieces of evidence to prove poisoning. OP agents form long-lived covalent reaction products (adducts) with endogenous proteins like human serum albumin (HSA) and butyrylcholinesterase (BChE). The adducted nonapeptide (NP) obtained by proteolysis of the BChE-adduct is one of the most sensitive and important biomarkers. We herein present a novel class of NP-adducts propionylated at its N-terminal phenylalanine residue (F195). The biomarker derivative is produced by addition of propionic anhydride to the NP-adduct inducing its quantitative conversion in aqueous buffer within 5 min at room temperature. Afterwards the mixture is directly analyzed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry (µLC-ESI MS/MS). The sensitivity of the method is comparable to that of the non-derivatized NP-adduct. These characteristics make the method highly beneficial for forensic analysis especially in cases in which the OP agent does not form adducts with HSA that are typically targeted as a second biomarker of exposure. This novel procedure was successfully applied to nerve agent-spiked samples sent by the Organisation for the Prohibition of Chemical Weapons (OPCW) as well as to plasma samples of real cases of pesticide poisoning.

Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study.

Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

Identification of creatine kinase and alpha-1 antitrypsin as protein targets of alkylation by sulfur mustard.

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide-thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys282 of CK and Cys232 of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.