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、灵敏的人体神经毒剂暴露测定方法。现有的检测方法不能检测到低剂量暴露，也不能向公众保证没有发生过暴露。血液中的两种蛋白质，丁基胆碱酯酶和白蛋白，将作为神经毒剂暴露的生物标志物在我们的新检测中进行测试。丁基胆碱酯酶是测定酶活性丧失的一种已建立的生物标志物。然而，测量丁基胆碱酯酶活性并不能确定抑制剂是一种神经毒剂，也没有显示低剂量暴露。白蛋白是一种新的生物标志物，它作为生物标志物的效用以前还没有在人类身上进行过测试，尽管它在老鼠身上表现出了希望。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附着在神经毒剂上的肽。选择性反应监测方法有望在人血浆中检测到少至100飞摩尔的共价结合神经毒剂。这些检测方法将在体外用索曼和沙林处理过的人血浆中开发。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估分析方法的效用。具体目标是：1)开发一种快速诊断神经毒剂暴露的方法，通过用索曼和沙林处理人体血液，并在质谱仪中分析样品。该方法将寻找白蛋白-神经毒剂加合物和丁基胆碱酯酶-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，通过检测意外接触有机磷农药的人的血液，或摄入有机磷农药的自杀未遂者的血液。质谱法将用于确定暴露程度。这一特定目标与目标1的不同之处在于，人类将暴露于神经毒剂模拟物中；神经毒剂模拟物必须进行研究，因为人类无法接触到神经毒剂。新的诊断方法有可能在紧急情况下挽救生命，因为正确、快速的诊断意味着适当的医疗。

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.