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

描述（由申请人提供）：原发性免疫缺陷疾病（PIDDs）是一大类免疫系统遗传疾病。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，没有可靠的筛查方法用于PIDDs的早期诊断。我们提案的目标是开发和验证一种特定的定量分析方法，该方法将使用少量血液同时识别多种pidd。我们之前开发了一种新的蛋白质组学筛选方法，使用选择性反应监测-质谱法（SRM-MS）同时鉴定来自跨膜蛋白簇分化3 （CD3）和细胞内蛋白，Wiskott-Aldrich综合征蛋白（WASP）和布鲁顿酪氨酸激酶（BTK）的低丰度特异性特征肽，作为三种危及生命的PIDDs的标志物；严重联合免疫缺陷（SCID）， Wiskott-Aldrich综合征（WAS）和x连锁阿甘球蛋白血症（XLA）。本应用程序的目的是通过开发肽免疫亲和富集与选定的反应监测-质谱（免疫- srm - ms）相结合的新方法来提高我们的新方法的灵敏度，以量化婴儿血液中的一组生物标志物，以促进多种危及生命的pidd的早期检测和诊断，并验证这些生物标志物的临床应用。我们的目标是：1。通过使用SRM-MS鉴定9种额外条件下的蛋白型特征肽，扩大现有的可筛选pidd面板。这些pidd包括ADA缺乏性SCID、MHC II类缺乏性SCID、高IgE复发性感染综合征、常见变异性免疫缺陷疾病（CVIDs） 3和8、共济失调毛细血管扩张症、噬血细胞性淋巴组织细胞增多症、x连锁淋巴增生性疾病和x连锁慢性肉芽肿病。我们将使用人类细胞系选择这些pidd的“特征”肽，并充分优化SRM-MS条件。2. 通过偶联肽免疫亲和富集，提高SRM-MS检测PIDDs的敏感性。我们将采用免疫srm程序来测量各种pidd的15个目标蛋白的特征肽，以提高我们的检测在临床实施中的灵敏度。我们将通过生成响应曲线来衡量每个检测的性能指标。3. 评估多重免疫- srm方法在更大的临床样本中正确识别特异性免疫缺陷患者的能力。我们的多重免疫- srm测定将用于西雅图儿童免疫诊断实验室回顾性和前瞻性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.