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

用于下一代新生儿威尔逊病、胱氨酸病和原发性免疫缺陷筛查的多重蛋白质组学分析

• 批准号: 10188579
• 负责人: Sihoun Hahn
• 金额: $ 58.64万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188579
• 关键词: Affect; Biological Assay; Blinded; Blood; Brain Injuries; Chemicals; Child; Chromatography; Complex Mixtures; Congenital Disorders; Cystinosis; Detection; Deuterium; Development; Diagnosis; Digestion; Disease; Early Diagnosis; Goals; Guidelines; Health Care Costs; Hepatolenticular Degeneration; Hereditary Disease; Hydrogen; Investigation; Isotopes; Kidney Failure; Laboratories; Lead; Left; Life; Liquid Chromatography; Liver Cirrhosis; Mass Spectrum Analysis; Measurable; Methods; Molecular; Monoclonal Antibodies; Mutation; Neonatal Screening; Newborn Infant; Organic solvent product; Patients; Peptides; Pilot Projects; Population; Procedures; Proteins; Proteomics; Protocols documentation; Reproducibility; Running; Sampling; Sepsis; Spottings; Testing; Time; Validation; Washington; base; congenital immunodeficiency; cost effective; disability; high throughput screening; improved outcome; interest; next generation; novel; performance tests; population based; protein biomarkers; sample collection; screening; tandem mass spectrometry; time use

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.

项目概要 新生儿胱氨酸病、威尔逊病 (WD) 和原发性免疫缺陷 (PIDD) 筛查至关重要， 因为尽管存在有效的治疗方法，但这些疾病仍然具有毁灭性和致命性。很遗憾， 大多数患有这些疾病的患者是在因缺乏药物而出现严重并发症后被诊断出来的。 具有成本效益的筛选方法。在出现这些负面后遗症之前接受治疗的患者 显着改善结果。许多先天性疾病是由突变引起的，这些突变导致缺失或缺失 蛋白质水平降低；因此，蛋白质生物标志物在诊断/筛查方面具有巨大的潜力 先天性疾病。作为概念验证，我们已经证明液相色谱法与 特征肽的串联质谱分析可以识别缺乏特定蛋白质标志物的患者 三种危及生命的 PIDD、胱氨酸病和 WD。我们相信新颖的靶向蛋白质组分析可以 用作快速、多重、廉价的方法来筛选各种可治疗的先天性 失调。我们的最终目标是将靶向蛋白质组学方法开发为适合的高通量筛选 轻松融入已建立的新生儿筛查工作流程和指南。我们的方法涉及直接量化 使用基于免疫亲和力的富集结合液体来提取极低丰度的蛋白质 色谱-串联质谱 (LC-MS/MS) 应用于新生儿筛查中的干血斑 牌。当前应用程序的目标是大幅减少该蛋白质组学的每个样本的计时器 分析，使其可用于基于人群的筛查。我们之前已经展示了以下能力 多重目标，可在单次运行中筛查单个患者的多种先天性疾病。我们的 中心假设是一种新的肽标记程序可以应用于蛋白质组工作流程 对多个患者进行多重分析，使其适合 NBS。我们的具体目标是： (1)：发展 新型肽标签和单克隆抗体中肽的特异性洗脱，以减少 每个样本的 NBS 运行时间。我们将开发使用一套新颖的肽标签，以便几个新生儿 可以在同一质谱运行中进行分析，以便多重患者样本并带来方法 每个新生儿的运行时间目标值 < 2 分钟。该蛋白质组方法优化的第二个组成部分 是利用富集后的单克隆抗体中的目标肽段进行特异性洗脱，进一步减少 样本复杂性和运行时间。 (2)：正常控制下的性能测试、优化和验证 样品。我们将检查的主要指标是新生儿内的再现性。第二个指标是 测量一组新生儿中非目标肽的干扰水平。 (3)：多维 对各种患者干血斑样本的盲法组进行多重验证。 (4)：试点研究 估计误报率。我们将在干燥的样品上测试完全优化的多重蛋白质组学方法 从华盛顿州国家统计局实验室获得的 10,000 名随机新生儿的血斑。