Multiplexed quantification of circulating peptidomic signatures for EBOLA early diagnosis

摘要 埃博拉病毒（Ebola virus，EBOV）是一种丝状的、有包膜的、非节段的负义RNA病毒，携带有病毒的RNA。 美国国立卫生研究院和疾病预防控制中心的A类优先病原体。感染EBOV会导致严重的出血热， 人类和非人类灵长类动物（NHP）的高死亡率。在2014年底西非爆发埃博拉疫情期间， 缺乏用于早期埃博拉病毒病（EVD）检测的实验室检测（最好在发病前）， 症状）和缺乏有效治疗限制了最终流行病的控制和管理。 为了满足EVD早期诊断的迫切需求，我们最近定制了一个平台， 增强生物标志物检测，以允许血清样品中灵敏和特异的EBOV生物标志物定量。 我们的方法使用抗体修饰的多孔硅纳米盘（pSiNDs）和高通量，高分辨率 质谱（MS）-统称为pSiND-MS -大大提高了特异性和灵敏度 从复杂的蛋白质样品中检测目标肽。在我们定制的EVD测定中，这些pSiND是 用针对EBOV基质蛋白VP 40的特异性肽的定制抗体进行功能化， 从EBOV感染宿主的非酶消化的血清样品中捕获这些肽。特别是 该测定的起始非酶消化步骤用于灭活EBOV颗粒和定位- 特异性消化所有血清蛋白用于随后的pSiND-MS分析。我们已经观察到VP 40肽 在感染动物的血浆中，甚至在EBOV RNA可检测到之前， 诊断分析我们已经鉴定了EBOV和EBOV物种特异性VP 40肽，并假设 我们选择的两种VP 40肽的多重检测将有力地指示EVD、EBOV种类和EBOV载量 在潜在感染者身上。因此，我们将应用pSiND-MS定量低丰度VP 40肽， EBOV的早期诊断，使用快速，单次测试测定，其中我们建议：1）优化pSiND-MS 用于检测这些生物标志物的平台和2）应用pSiND-MS纵向定量血清中的VP 40水平 从美国陆军传染病医学研究所感染EBOV的非人类灵长类动物， 产生信息性的临床前EBOV检测谱。 我们的方法的主要优点包括样品制备的简单性和安全性;基质的使用- 辅助激光解吸/电离飞行时间（MALDI-TOF）MS设备在大多数医院都有， 实验室（以及未来使用简化的小型化MS系统）;在MS/MS上读取分析的潜力 用于标记序列确认的仪器;生物标记检测的特异性和灵敏度; 可快速定制的模块化多重测定;和快速诊断（从样品开始2小时）。这些特点 非常适合即时应用。该方案的成功完成将提供一种新的技术 它可以克服目前埃博拉和其他传染性病原体诊断方法的重大障碍， 可能改变护理、治疗和遏制未来流行病的方案。

ABSTRACT Ebola virus (EBOV) is classified as a filamentous, enveloped, non-segmented negative-sense RNA carrying Category A Priority Pathogen by the NIH and CDC. Infections with EBOV cause severe hemorrhagic fever and high mortality in humans and non-human primates (NHPs). During the Ebola outbreak of late 2014 in West Africa, the lack of laboratory tests for early Ebola-virus-disease (EVD) detection (preferably before the onset of symptoms) and lack of effective treatments limited the containment and management of the eventual epidemic. To address this critical need for early EVD diagnosis, we have recently customized a platform we developed to enhance biomarker detection to allow sensitive and specific EBOV biomarker quantification in serum samples. Our method uses antibody-modified porous silicon nanodisks (pSiNDs) and high-throughput, high-resolution mass spectrometry (MS) – together referred to as pSiND-MS – to greatly enhance the specificity and sensitivity of target peptide detection from complex protein samples. In our customized EVD assay, these pSiNDs are functionalized with custom antibodies to specific peptides of the EBOV matrix protein VP40 in order to efficiently capture these peptides from non-enzymatically-digested serum samples of EBOV-infected hosts. Notably, the starting non-enzymatic digestion step of this assay is used to both inactivate live EBOV particles and to site- specifically digest all serum proteins for subsequent pSiND-MS analysis. We have observed that VP40 peptides are increased in the plasma of infected animals even before EBOV RNA is detectable with currently available diagnostic assays. We have identified EBOV- and EBOV-species-specific VP40 peptides, and hypothesize that multiplex detection of our two selected VP40 peptides will robustly indicate EVD, EBOV species and EBOV load in potentially-infected individuals. We will thus apply pSiND-MS to quantify low-abundance VP40 peptides for early diagnosis of EBOV using a rapid, single-test assay, where we propose to: 1) optimize the pSiND-MS platform for detection of these biomarkers and 2) apply pSiND-MS to longitudinally quantify VP40 levels in sera from EBOV-infected non-human primates at the U.S. Army Medical Research Institute of Infectious Diseases, to produce an informative preclinical EBOV-detection profile. Major advantages of our approach include the simplicity and safety of sample preparation; the use of matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS equipment found in most hospitals and laboratories (and future use of simplified, miniaturized MS systems); the potential to read the assay on MS/MS instruments for marker sequence confirmation; the specificity and sensitivity of biomarker detection; the use of rapidly-customizable, modular multiplex assay; and rapid diagnosis (2hrs from sample). These characteristics are ideal for a point-of-need application. Successful completion of this proposal will provide a novel technology that can overcome significant hurdles of current diagnostic methodologies for Ebola and other infectious agents, potentially transforming the protocols for care, treatment, and containment of future epidemics.