Multiplex Proteomic Analysis for Next-Generation Newborn Screening of Wilson Disease, Cystinosis, and Primary Immunodeficiencies

项目摘要 新生儿筛查胱氨酸病、威尔逊病（WD）和原发性免疫缺陷（PIDD）至关重要， 因为这些疾病是毁灭性的和致命的，尽管存在有效的治疗方法。不幸的是， 大多数患有这些疾病的患者是在发展出严重的并发症后被诊断出来的， 具有成本效益的筛查方法。在这些阴性后遗症发作前接受治疗的患者， 显著改善的结果。许多先天性疾病是由突变引起的， 蛋白质水平降低;因此，蛋白质生物标志物在诊断/筛选 先天性疾病作为概念验证，我们已经证明，液相色谱结合 特征肽的串联质谱分析可以鉴定缺乏特异性蛋白质标志物的患者 三种危及生命的PIDD胱氨酸病和WD我们相信，新的靶向蛋白质组学分析可以 作为一种快速、多路复用、廉价的方法，用于筛查各种可治疗的先天性 紊乱我们的最终目标是将靶向蛋白质组学方法发展成适合的高通量筛选 容易纳入既定的新生儿筛查工作流程和指南。我们的方法包括直接量化 使用基于免疫亲和性的富集结合液体 色谱-串联质谱（LC-MS/MS）在新生儿筛查中的应用 卡.本申请的目的是大大减少这种蛋白质组学的每个样本的时间 分析，以便它可以用于基于人口的筛选。我们之前已经证明了 多重靶标，用于在单次运行中筛查单个患者的多种先天性疾病。我们 中心假设是，一种新的肽标记程序可以应用于蛋白质组学工作流程， 多路复用用于多个患者，使得其适合于NBS。我们的具体目标是：（1）：发展 新的肽标签和从单克隆抗体中特异性洗脱肽， 每个样本的NBS运行时间。我们将开发一套新的肽标签的使用， 可以在同一质谱运行中进行分析，以多重患者样本并带来方法 运行时间降低到每名新生儿< 2分钟的目标值。这种蛋白质组学方法优化的第二个组成部分是 是使用从富集后的单克隆抗体中特异性洗脱靶肽，以进一步降低 示例复杂性和运行时间。(2)：正常控制中的性能测试、优化和验证 样品我们将检查的主要指标是新生儿内的可重复性。第二个指标是 在一组新生儿中测量非靶肽的干扰水平。(3)：多维度 在各种患者干血斑样品的盲组中进行多重验证。(4)：试点研究， 估计假阳性率。我们将在干细胞上测试完全优化的多重蛋白质组学方法。 从华盛顿国家统计局实验室随机抽取10，000名新生儿的血斑。

Project Summary Newborn screening for Cystinosis, Wilson disease (WD) and primary immunodeficiencies (PIDD) is critical, because these disorders are devastating and fatal despite the existence of validated treatments. Unfortunately, most patients with these disorders are diagnosed after developing significant complications because of a lack of cost-effective screening methods. Patients treated before the onset of these negative sequalae have significantly improved outcomes. Many congenital disorders are caused by mutations that result in absent or diminished levels of proteins; thus, protein biomarkers have enormous potential in the diagnosis/screening of congenital disorders. As proof-of-concept, we have demonstrated that liquid chromatography combined with tandem mass spectrometry analysis of signature peptides can identify patients lacking specific protein markers of three life-threatening PIDD, cystinosis and WD. We believe that novel targeted proteomic analyses can be utilized as rapid, multiplexed, inexpensive approaches to screen for a broad variety of treatable congenital disorders. Our ultimate goal is to develop targeted proteomic methods into high-throughput screens that fit readily into established newborn screening workflows and guidelines. Our methods involve direct quantification of extremely low abundance proteins using immuno-affinity-based enrichment combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) applied to dried blood spots on newborn screening cards. The objective of the current application is to drastically reduce the timer-per-sample for this proteomic analysis so that it can be used for population-based screening. We have previously demonstrated the ability to multiplex targets for the screening of numerous congenital disorders in a single patient in a single run. Our central hypothesis is that a novel peptide tagging procedure can be applied to the proteomic workflow for the multiplexing for multiple patients so that it is appropriate for NBS. Our specific aims are: (1): Development of novel peptide tags and specific elution of peptides from monoclonal antibodies in order to reduce the NBS run-time per sample. We will develop the use of a novel set of peptide tags so that several newborns can be analyzed in the same mass spectrometry run in order to multiplex patient samples and bring method runtimes to a target value of < 2 min per newborn. A second component of this proteomic method optimization is the use of specific elution of target peptides from monoclonal antibodies after enrichment to further reduce sample complexity and runtime. (2): Performance testing, optimization and validation in normal control samples. The main metric that we will examine is the within-newborn reproducibility. The second metric is to gauge the level of interference from non-target peptides across a panel of newborns. (3): Multi-dimensional multiplex validation in blinded sets of various patient dried blood spot samples. (4): Pilot study to estimate the rate of false positives. We will test the fully optimized multiplex proteomic method on dried blood spots from 10,000 random newborns obtained from the Washington State NBS laboratory.