Peptide Fingerprint multiplex analysis by MS/MS for primary immundeficiency

描述(由申请人提供)：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)是三种主要的免疫缺陷疾病，如果诊断和治疗不当，会导致早期死亡。然而，早期识别、诊断和先发制人的治疗对于改善这些疾病的预后至关重要；我们有能力进行基于人群的筛查，而不仅仅是SCID。长期目标是开发一种可行的多重检测方法，使之能够同时识别以缺乏特定蛋白质为特征的多种先天性疾病，方法是使用少量干燥到滤纸上的血液样本。这一特殊应用的目的是识别和表征可用于快速识别三种严重免疫缺陷综合征患者的独特“指纹”多肽，这三种严重免疫缺陷综合征将被用作模型疾病：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)。将使用干血斑作为底物，检测方法将采用串联质谱仪。该应用的中心假设是串联质谱仪可用于检测来自CD3、WASP和BTK(分别用于SCID、WAS和XLA的标记)的关键“指纹”多肽的存在或不存在。这一假设是基于我们的初步数据(DeWilde等人，2008年)提出的。这项拟议研究的基本原理是，对严重的原发免疫缺陷疾病，如SCID、WAS和XLA的快速、早期检测将允许先发制人的治疗，从而防止死亡和发病率，从而提高生活质量和长期生产力。核心假设将通过追求三个具体目标来检验：1)识别CD3、WASP和BTK的关键“指纹”多肽；标记SCID的蛋白质。和XLA。2.)确定用于串联质谱仪(MS/MS)的每个预测的“指纹”多肽的适用性，并优化其检测。3.)验证每个“指纹”肽在SDIC、WAS、XLA患者和对照组的干血斑点中识别CD3、WASP和BTK缺失的能力。这种方法是创新的，因为我们开发了新的方法，扩大了现有技术的使用，以诊断由于缺乏特定蛋白质而导致的先天性疾病。这些工具有可能提高我们早期和快速识别几种严重的原发免疫缺陷疾病的能力。这项拟议的研究意义重大，因为它将提供一种测试方法，既可以用于新生儿筛查，也可以用于这些和其他严重的免疫缺陷和先天性疾病的快速诊断。这将提供机会在生命早期识别患者，并提供挽救生命的疗法。此外，预计这将从根本上促进蛋白质组学方法在诊断测试中的使用，这一领域前景广阔，但在很大程度上尚未开发。 公共卫生相关性：拟议的研究重点是早期和快速诊断严重的、危及生命的免疫缺陷综合征严重联合免疫缺陷(SCID)、Wiskott-Aldrich综合征(Was)和X连锁无丙种球蛋白血症(XLA)。该应用程序旨在利用一种创新的串联质谱学方法，使用干燥到滤纸上的血点来快速诊断这些疾病，由于该方法的高通量、多重潜力，它将极大地扩展我们使用非常小的血量对这些疾病和其他疾病进行新生儿筛查和快速诊断测试的能力。这将为危及生命的免疫缺陷疾病提供早期识别和先发制人治疗的机会，从而直接影响患者的护理。

DESCRIPTION (provided by applicant): Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and X-Linked Agammaglobulinemia (XLA) are three primary immune deficiency disorders associated with early lethality if not diagnosed and treated appropriately. Early recognition, diagnosis, and preemptive treatment are critical for improving outcomes in these disorders however; there is a fundamental gad in our ability to perform population-based screening for more than just SCID. The long-term goal is to develop a viable multiplex testing methodology that will allow simultaneous identification of multiple congenital disorders characterized by absence of specific proteins using small sample volumes of blood dried onto filter paper. The objective of this particular application is to identify and characterize unique "fingerprints" peptides that can be used to rapidly identify patients with three severe immunodeficiency syndromes that will be used as model disorders: Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and X-Linked Agammaglobulinemia (XLA). Dried blood spots will be used as the substrate and the testing approach will be tandem mass spectrometry. The central hypothesis of this application is that tandem mass spectrometry can used to detect the presence or absence of key "fingerprint" peptides derived from CD3, WASP and BTK (markers for SCID, WAS, and XLA respectively). This hypothesis has been formulated based on our preliminary data (deWilde et.al., 2008). The rationale for the proposed research is that rapid, early detection of severe primary immunodeficiency disorder such as SCID, WAS, and XLA will allow for preemptive treatment that will prevent death and morbidity thereby improving quality of life and long-term productivity. The central hypothesis will be tested by pursuing three specific aims: 1.) Identify key "fingerprint" peptides for CD3, WASP, and BTK; marke proteins for SCID, WAS. And XLA respectively. 2.) Define the suitability of each predicted "fingerprint" peptide for use in tandem mass spectrometry (MS/MS) and optimize its detection. 3.) Validate the ability of each "fingerprint" peptide to identify the absence of CD3, WASP, and BTK in dried blood spots from patients with SDIC, WAS, and XLA as well as controls. The approach is innovative because of the new methods we have developed, expanding the use of an existing technology to diagnose congenital disorders that result from absence of a specific protein. These tools have the potential to increase our capacity for early and rapid identification of several severe primary immunodeficiency disorders. The proposed research is significant, because it will provide a testing methodology that is viable both for newborn screening and for rapid diagnostics for these and other severe immunodeficiency and congenital disorders. This will provide opportunities to identify patients early in life and provide life-saving therapies. In addition, this is expected to fundamentally advance the use of proteomic methodologies for diagnostic testing, an area that holds great promise but is largely untapped. PUBLIC HEALTH RELEVANCE: The proposed studies focus on early and rapid diagnosis of the severe, life-threatening immunodeficiency syndromes Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and X-Linked Agammaglobulinemia (XLA). This application is designed to utilize an innovative tandem mass spectroscopy approach to quickly diagnose these disorders using blood spots dried onto filter paper, because of the high-throughput, multiplex potential of this method, it will dramatically expand our ability to perform newborn screening and rapid diagnostic testing for these and other disorders using very small blood volumes. This will directly impact patient care by providing early identification and the opportunity for preemptive treatment for life threatening immunodeficiency disorders.