Multiplexed immuno-SRM screening for primary immunodeficiencies

 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。目前治疗SCID的方法是利用定量聚合酶链式反应来检测T细胞受体切除环(TREC)，这只适用于免疫缺陷的一部分。我们建议的目标是开发和验证一种特定的定量检测方法，该方法将使用干血迹(DBS)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)的特定标志肽和细胞内蛋白，即威斯科特-阿尔德里奇综合征蛋白(Wasp)和布鲁顿酪氨酸激酶(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连锁无丙种球蛋白血症(XLA)。这项应用的目的是提高我们新方法的灵敏度，通过开发多肽免疫亲和富集与SRM-MS(免疫-SRM-MS)相结合来定量DBS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。我们的目标是：1.通过使用SRM-MS鉴定8种额外情况下的蛋白质型特征多肽，扩大现有的可筛选PIDD的范围。这些PIDD包括ADA缺陷SCID、MHC II类缺陷SCID、DOCK8缺陷、常见变量免疫缺陷、毛细血管扩张性共济失调、家族性噬血细胞淋巴组织细胞增生症2、X连锁淋巴增殖综合征和X连锁慢性肉芽肿性疾病。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DBS中目标蛋白的标志肽，以提高我们检测的灵敏度。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方法在大量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分析将在西雅图儿童免疫诊断实验室收集的患者DBS样本上进行测试。将检索和检测来自佤邦的阳性新生儿DBS。

 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. The current approach to SCID utilizes quantitative PCR to detect T cell Receptor Excision Circles (TREC) which is applicable only to a subset of immunodeficiencies. The goal of our proposal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple PIDDs using dried blood spots (DBS). We previously developed a novel proteomic screening method using Selected Reaction Monitoring-Mass Spectrometry (SRM-MS) to simultaneously identify 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 SRM-MS (immuno-SRM-MS) to quantify a panel of biomarkers in DBS to facilitate the early detection and diagnosis of multiple life-threatening PIDDs. Our Aims are to: 1. Expand the existing panel of screenable PIDDs by identifying proteotypic signature peptides for 8 additional conditions using SRM-MS. These PIDDs include ADA-deficient SCID, MHC class II deficient SCID, DOCK8 deficiency, Common Variable Immunodeficiency, Ataxia Telangiectasia, Familial hemophagocytic lymphohistiocytosis 2, X-linked lymphoproliferative syndrome, 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 target proteins in DBS for various PIDDs to improve the sensitivity of our assay. 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 large set of clinical samples. Our multiplexed immuno-SRM assay will be tested on patient DBS samples collected by the Seattle Children's Immunology Diagnostic Laboratory. Positive newborn DBS from WA State will be retrieved and tested.