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

项目摘要 新生儿膀胱炎、威尔逊病(WD)和原发免疫缺陷(PIDD)筛查至关重要， 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出现严重并发症后被诊断出来的，因为缺乏 高性价比的筛查方法。在这些阴性后遗症发生之前接受治疗的患者 显著改善了结果。许多先天性疾病是由导致缺失或缺失的突变引起的。 蛋白质水平降低；因此，蛋白质生物标记物在诊断/筛查糖尿病方面具有巨大潜力。 先天性疾病。作为概念验证，我们已经证明了将高效液相色谱与 串联质谱学分析标志性多肽可以识别缺乏特定蛋白标志物的患者 三个危及生命的PIDD，胱氨酸病和WD。我们相信新的有针对性的蛋白质组分析可以 作为快速、多元、廉价的方法用于筛查各种可治疗的先天性心脏病 精神错乱。我们的最终目标是将有针对性的蛋白质组学方法开发成符合以下条件的高通量筛选 可随时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 免疫亲和结合液体法分离极低丰度蛋白质 气相色谱-串联质谱仪(LC-MS/MS)在新生儿血斑筛查中的应用 扑克牌。目前应用的目标是大幅减少这种蛋白质组的每个样本的计时器 分析，以便它可以用于基于人群的筛查。我们之前已经证明了有能力 在一次运行中对单个患者中的众多先天性疾病进行筛查的多个目标。我们的 中心假设是一种新的肽标签程序可以应用于蛋白质组的工作流程 对多个患者进行多路复用，以便适合于NBS。我们的具体目标是：(1)发展 新的多肽标签和从单抗中特异性洗脱多肽以减少 每个样本的国家统计局运行时间。我们将开发一套新的多肽标签的用途，以便几个新生儿 可以在同一次质谱分析中运行，以便对患者样本进行多重分析并带来方法 运行时间为每个新生儿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 是利用特异的洗脱法从单抗中洗脱出目标多肽，经过浓缩后进一步减少 样本复杂性和运行时。(2)：正常控制下的性能测试、优化和验证 样本。我们将检查的主要指标是新生儿体内的可重复性。第二个指标是 测量非靶标多肽对一组新生儿的干扰程度。(3)：多维性 不同患者干血斑样本盲集的多重验证。(4)：试点研究 估计误报的比率。我们将对完全优化的复合蛋白质组学方法进行测试 从华盛顿州国家统计局实验室随机采集的1万名新生儿的血迹。

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.