Mass Spectrometry in Clinical Diagnosis of Nerve Agent Exposure

描述（由申请人提供）：本项目的目标是开发一种快速、灵敏的人体神经毒剂暴露检测方法。现有的检测方法不能检测低剂量暴露，也不能向公众保证没有发生暴露。血液中的两种蛋白质，丁酰胆碱酯酶和白蛋白，将在我们的新检测中作为神经毒剂暴露的生物标志物进行测试。丁酰胆碱酯酶是测定酶活性损失的测定中确定的生物标志物。然而，丁酰胆碱酯酶活性的测量并不能确定这种抑制剂是神经毒剂，也不能揭示低剂量接触。白蛋白是一种新的生物标志物，其作为生物标志物的效用以前没有在人类中进行过测试，尽管它在小鼠中显示出希望。我们提出的新检测方法使用MALDI-TOF和串联质谱法来检测共价连接到神经毒剂的肽。选择性反应监测方法预计可检测到人体血浆中低至100飞摩尔的共价结合神经毒剂。将在体外用梭曼和沙林处理过的人血浆进行测定。将通过检测暴露于神经毒剂模拟物的人类受试者的血浆来评价试验的实用性。具体目标是：1）通过用梭曼和沙林处理人体血液，并在质谱仪中分析样本，开发一种快速诊断神经毒剂暴露的方法。该方法将寻找白蛋白-神经毒剂加合物和丁酰胆碱酯酶-神经毒剂加合物的肽。2)开发一种快速诊断神经毒剂模拟物暴露的方法，通过检测意外暴露于有机磷农药的人或摄入有机磷农药的自杀未遂病例的血液。质谱法将用于识别暴露。这一具体目标与目标1的不同之处在于，人类将暴露于神经毒剂模拟物;必须研究神经毒剂模拟物，因为暴露于神经毒剂的人类不可用。新的诊断方法具有在紧急情况下挽救生命的潜力，因为正确、快速的诊断表明了适当的治疗。

DESCRIPTION (provided by applicant): The goal of this project is to develop a rapid and sensitive assay for nerve agent exposure in humans. Existing assays do not detect low dose exposure and cannot reassure the public that no exposure has occurred. Two proteins in blood, butyrylcholinesterase and albumin, will be tested as biomarkers of nerve agent exposure in our new assays. Butyrylcholinesterase is an established biomarker in assays that measure loss of enzyme activity. However, measurement of butyrylcholinesterase activity does not identify the inhibitor as a nerve agent, and does not reveal low dose exposure. Albumin is a new biomarker whose utility as a biomarker has not previously been tested in humans, though it shows promise in mice. Our proposed new assays use MALDI-TOF and tandem mass spectrometry to detect peptides covalently attached to nerve agent. The Selective Reaction Monitoring method is expected to detect as little as 100 femtomoles of covalently bound nerve agent in human plasma. The assays will be developed with human plasma treated with soman and sarin in vitro. The utility of the assays will be evaluated by testing plasma from human subjects exposed to nerve agent simulants. The specific aims are 1) Develop a method to rapidly diagnose exposure to nerve agents, by treating human blood with soman and sarin, and analyzing samples in the mass spectrometer. The method will look for peptides of albumin-nerve agent adducts and of butyrylcholinesterase-nerve agent adducts. 2) Develop a method to rapidly diagnose exposure to nerve agent simulants, by testing blood from humans accidentally exposed to organophosphorus pesticides, or from attempted suicide cases who ingested organophosphorus pesticides. Mass spectrometry will be used to identify exposure. This specific aim differs from aim #1 in that humans will have been exposed to nerve agent simulants; nerve agent simulants must be studied because humans exposed to nerve agents are not available. The new diagnostic methods have the potential for saving lives in an emergency because correct, rapid diagnosis indicates the appropriate medical treatment.