Peptide Fingerprint multiplex analysis by MS/MS for primary immundeficiency

描述（由申请方提供）：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）是三种主要免疫缺陷疾病，如果未得到适当诊断和治疗，则与早期致死性相关。早期识别、诊断和抢先治疗对于改善这些疾病的预后至关重要;然而，我们有能力进行以人群为基础的筛查，而不仅仅是SCID。 长期目标是开发一种可行的多重检测方法，该方法将允许使用干燥到滤纸上的少量血液样本同时鉴定以缺乏特定蛋白质为特征的多种先天性疾病。本特定申请的目的是鉴定和表征独特的“指纹”肽，其可用于快速鉴定具有三种严重免疫缺陷综合征的患者，所述三种严重免疫缺陷综合征将用作模型病症：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，检测方法为串联质谱法。本申请的中心假设是串联质谱法可用于检测源自CD 3、WASP和BTK（分别为SCID、WAS和XLA的标志物）的关键“指纹”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）。拟议研究的基本原理是，快速，早期发现严重的原发性免疫缺陷疾病，如SCID，WAS和XLA将允许先发制人的治疗，这将防止死亡和发病，从而提高生活质量和长期生产力。中心假设将通过追求三个具体目标进行测试：1。识别CD 3、WASP和BTK的关键“指纹”肽; SCID、WAS的马克蛋白。分别为XLA。2.）的情况。定义每个预测的“指纹”肽在串联质谱法（MS/MS）中使用的适用性并优化其检测。3.）第三章验证每种“指纹”肽鉴定SDIC、WAS和XLA患者以及对照的干血斑中不存在CD 3、WASP和BTK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，以诊断因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性免疫缺陷疾病的能力。拟议的研究是重要的，因为它将提供一个测试方法，是可行的新生儿筛查和快速诊断这些和其他严重的免疫缺陷和先天性疾病。这将为在生命早期识别患者并提供挽救生命的治疗提供机会。此外，预计这将从根本上推进蛋白质组学方法用于诊断测试的使用，这是一个有很大希望但基本上尚未开发的领域。 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）。该应用旨在利用创新的串联质谱方法，使用滤纸上干燥的血点快速诊断这些疾病，由于该方法的高通量，多重潜力，它将大大扩展我们使用非常小的血液体积对这些和其他疾病进行新生儿筛查和快速诊断测试的能力。这将通过提供早期识别和对危及生命的免疫缺陷疾病进行先发制人治疗的机会来直接影响患者护理。

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.