Multiplexed immuno-SRM screening for primary immunodeficiencies

 描述（由申请人提供）：原发性免疫缺陷疾病（PIDD）是一大类免疫系统遗传性疾病。这些疾病的严重程度和症状范围各不相同，但如果没有有效的早期治疗，它们可能是致命的。目前的 SCID 方法利用定量 PCR 来检测 T 细胞受体切除环 (TREC)，该方法仅适用于免疫缺陷的一部分。我们提案的目标是开发并验证一种特定的定量检测方法，该检测方法将使用干血斑 (DBS) 同时识别多个 PIDD。我们之前开发了一种新型蛋白质组筛选方法，使用选择反应监测质谱 (SRM-MS)，同时鉴定源自分化跨膜蛋白簇 3 (CD3) 的特定特征肽和细胞内蛋白、Wiskott-Aldrich 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 的标记物；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)。本应用的目的是通过开发与 SRM-MS (immuno-SRM-MS) 结合的肽免疫亲和富集来量化 DBS 中的一组生物标志物，从而提高我们新方法的灵敏度，从而促进多种危及生命的 PIDD 的早期检测和诊断。我们的目标是： 1. 通过使用 SRM-MS 识别 8 个附加条件的蛋白型特征肽，扩展现有的可筛选 PIDD 组。这些 PIDD 包括 ADA 缺陷 SCID、MHC II 类缺陷 SCID、DOCK8 缺陷、常见变异性免疫缺陷、共济失调性毛细血管扩张症、家族性噬血细胞性淋巴组织细胞增多症 2、X 连锁淋巴细胞增殖综合征和 X 连锁慢性肉芽肿病。我们将使用人类细胞系来选择这些 PIDD 的“特征”肽，并全面优化 SRM-MS 条件。 2. 通过与肽免疫亲和富集结合，提高 SRM-MS 检测 PIDD 的灵敏度。我们将采用免疫 SRM 程序来测量 DBS 中针对各种 PIDD 的靶蛋白的特征肽，以提高我们测定的灵敏度。我们将通过生成响应曲线来测量每个测定的性能指标。 3. 评估多重免疫 SRM 方法在大量临床样本中正确识别患有特定免疫缺陷的患者的能力。我们的多重免疫 SRM 检测将在西雅图儿童免疫学诊断实验室收集的患者 DBS 样本上进行测试。来自西澳州的阳性新生儿 DBS 将被取出并进行检测。

 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.