Mass Spectrometry in Clinical Diagnosis of Nerve Agent Exposure

描述(由申请人提供)：该项目的目标是开发一种快速而灵敏的人类神经毒剂暴露检测方法。现有的检测方法不能检测到低剂量的接触，也不能向公众保证没有发生过接触。在我们的新检测中，血液中的两种蛋白质，丁酰胆碱酯酶和白蛋白，将被测试为神经毒剂暴露的生物标志物。丁酰胆碱酯酶是一种公认的生物标志物，用于衡量酶活性的丧失。然而，丁酰胆碱酯酶活性的测量没有确定该抑制剂是一种神经毒剂，也没有发现低剂量暴露。白蛋白是一种新的生物标记物，其作为生物标记物的效用以前从未在人类身上进行过测试，尽管它在小鼠身上显示出了希望。我们建议的新分析方法使用MALDI-TOF和串联质谱仪来检测与神经毒剂共价连接的多肽。选择性反应监测方法预计可以检测到人体血浆中低至100个毫微分子的共价结合神经毒剂。这些检测方法将在体外用梭曼和沙林处理的人血浆进行开发。这些检测的有效性将通过测试暴露于神经毒剂模拟物的人体受试者的血浆来评估。具体目标是1)开发一种快速诊断暴露于神经毒剂的方法，方法是用梭曼和沙林处理人血，并在质谱仪中分析样本。该方法将寻找白蛋白-神经毒剂加合物和丁酰胆碱酯酶-神经毒剂加合物的多肽。2)开发一种快速诊断暴露于神经毒剂模拟物的方法，方法是检测意外接触有机磷杀虫剂的人的血液，或摄入有机磷杀虫剂的自杀未遂病例的血液。质谱学将被用来识别暴露。这一具体目标与目标1的不同之处在于，人类将暴露在神经毒剂模拟物中；必须研究神经毒剂模拟物，因为无法获得暴露于神经毒剂的人类。新的诊断方法有可能在紧急情况下拯救生命，因为正确、快速的诊断表明适当的医疗治疗。

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.