title: A liquid chromatography tandem mass spectrometric method on in vitro nerve agents poisoning characterization and reactivator efficacy evaluation by determination of specific peptide adducts in acetylcholinesterase.
abstract: The terroristic availability of highly toxic nerve agents (NAs) highlights the necessity for a deep understanding of their toxicities and effective medical treatments. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for a characterization of the NAs poisoning and an evaluation on the efficacy of reactivators in in vitro was developed for the first time. After exposure to sarin or VX and pepsin digestion, the specific peptides of acetylcholinesterase (AChE) in a purified status, i.e. undecapeptide "GESAGAASVGM" in free, unaged, or aged status was identified and quantified. A key termination procedure is focused to make the reaction system "frozen" and precisely "capture" the poisoning, aging and spontaneous reactivation status of AChE, and the abundance of such specific peptides can thus be simultaneously measured. In our established method, as low as 0.72% and 0.84% inhibition level of AChE induced by 0.5nM sarin and VX can be detected from the measurement of peptide adducts, which benefits a confirmation of NAs exposure, especially at extremely low levels. Comparing with conventional colorimetric Ellman assays, our method provides not only enzyme activity and inhibition rate, but also the precise poisoning status of NAs exposed AChE. Based on the full information provided by this method, the efficacy of reactivators, such as HI-6, obidoxime and pralidoxime, in the typical treatment of NAs poisoned AChE in in vitro was further evaluated. Our results showed that this method is a promising tool for the characterization of NAs poisoning and the evaluation of reactivator efficacy.
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China.
authors.forename: Long
authors.initials: L
authors.lastname: Yan
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China. Electronic address: tangjijun2012@163.com.
authors.forename: Jijun
authors.initials: J
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China. Electronic address: xiejw@bmi.ac.cn.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Xie
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology / Q000506:poisoning; D002801:Cholinesterase Reactivators / Q000494:pharmacology*; D002853:Chromatography, Liquid; D003124:Colorimetry; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D000067397:Nerve Agents / Q000737:chemistry / Q000494:pharmacology* / Q000506:poisoning*; D009768:Obidoxime Chloride / Q000494:pharmacology; D010091:Oximes / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011220:Pralidoxime Compounds / Q000494:pharmacology; D011726:Pyridinium Compounds / Q000494:pharmacology; D012524:Sarin / Q000737:chemistry / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D000067397:Nerve Agents; D010091:Oximes; D010446:Peptide Fragments; D011220:Pralidoxime Compounds; D011726:Pyridinium Compounds; D009768:Obidoxime Chloride; D012524:Sarin; D000110:Acetylcholinesterase; C022870:asoxime chloride; C028797:pralidoxime
keywords: Acetylcholinesterase; LC–MS/MS; Nerve agents; Peptide adduct; Reactivator