Multiplex Test for Primary Immunodeficiencies by Affinity Column coupled to MS/MS

描述(申请人提供)：原发免疫缺陷疾病(PIDD)是免疫系统的一大类遗传性疾病。这些疾病的严重程度和症状范围各不相同，但如果没有有效和早期的治疗，它们可能是致命的。目前，尚无可靠的筛查方法用于PIDD的早期诊断。我们建议的目标是开发和验证一种特定的定量检测方法，该方法将使用少量血液同时识别多个PIDD。我们先前开发了一种新的蛋白质组筛选方法，使用选择性反应监测-质谱仪同时鉴定来自跨膜分化蛋白簇3(CD3�)的低丰度特异性特征多肽以及细胞内蛋白，Wiskott-Aldrich综合征蛋白(Wasp)和Bruton酪氨酸激酶(BTK)作为三种危及生命的PIDD的标志物；严重联合免疫缺陷(SCID)、Wiskott-Aldrich综合征(WAS)和X连锁无镁球蛋白血症(XLA)。这项应用的目的是提高我们新方法的灵敏度，方法是开发多肽免疫亲和富集与选择性反应监测-质谱学(免疫-SRM-MS)相结合的方法，以量化婴儿血液中的一组生物标记物，以便于早期检测和诊断多个危及生命的PIDD，并验证这些生物标记物组的临床实施。我们的目标是：1.通过使用SRM-MS鉴定9种额外情况的蛋白质型标志性多肽，扩大现有的可筛选PIDD的小组。这些PIDD包括ADA缺陷的SCID、MHC II类缺陷的SCID、高IgE复发感染综合征、2种常见的可变免疫缺陷疾病(CVID)3和8、毛细血管扩张症、噬血细胞淋巴组织细胞增生症、X连锁淋巴组织增生性疾病和X连锁慢性肉芽肿疾病。我们将使用人类细胞系为这些PIDD选择“标志性”多肽，并充分优化SRM-MS条件。2.通过将SRM-MS与多肽免疫亲和富集结合起来，提高了SRM-MS检测PIDDS的灵敏度。我们将使用免疫-SRM程序来测量不同PIDD的15个目标蛋白的标志肽，以提高我们的检测方法的灵敏度，用于临床实施。我们将通过生成响应曲线来测量每个测试的性能指标。3.评估多重免疫-SRM方法在更大的临床样本中正确识别特定免疫缺陷患者的能力。我们的多重免疫-SRM分析将应用于西雅图儿童免疫诊断实验室回溯和预期收集的人类临床样本。

DESCRIPTION (provided by applicant): Primary immunodeficiency diseases (PIDDs) are a large group of genetic disorders of the immune system. These disorders vary in the severity and spectrum of symptoms, but without effective and early treatment, they can be fatal. Currently, there is no reliable screening assay for early diagnosis of PIDDs. The goal of our proposal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple PIDDs using a small volume of blood. We previously developed a novel proteomic screening method using Selected Reaction Monitoring-Mass Spectrometry (SRM-MS) to simultaneously identify low-abundant specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3�) and the intracellular proteins, Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and X-linked Agammaglobulinemia (XLA). The objective of this application is to improve the sensitivity of our novel approach by developing peptide immunoaffinity enrichment coupled to selected reaction monitoring- mass spectrometry (immuno-SRM-MS) to quantify a panel of biomarkers in infant blood to facilitate the early detection and diagnosis of multiple life-threatening PIDDs and validate these panels of biomarkers for clinical implementation. Our Aims are to: 1. Expand the existing panel of screenable PIDDs by identifying proteotypic signature peptides for 9 additional conditions using SRM-MS. These PIDDs include ADA- deficient SCID, MHC class II deficient SCID, Hyper IgE recurrent infection syndrome, 2 Common Variable Immunodeficiency Disorders (CVIDs) 3 and 8 , Ataxia Telangiectasia, Hemophagocytic lymphohistiocytosis, X-linked lymphoproliferative disease, and X-linked chronic granulomatous disease. We will use human cell lines to select "signature" peptides for these PIDDs and fully optimize SRM-MS conditions. 2. Increase sensitivity of the SRM-MS assay for PIDDs by coupling it with peptide immunoaffinity enrichment. We will employ immuno-SRM procedures for measurements of signature peptides for the 15 target proteins for various PIDDs to improve the sensitivity of our assay for clinical implementation. We will measure performance metrics for each assay by generating a response curve. 3. Evaluate the ability of a multiplex immuno-SRM approach to correctly identify patients with specific immunodeficiencies in a larger set of clinical samples. Our multiplex immuno-SRM assay will be deployed on human clinical samples retrospectively and prospectively collected by the Seattle Children's Immunology Diagnostic Laboratory.