Peptide Immunoaffinity enriched LC-MRM-MS analysis for Cystinosis/Wilson disease

描述（由申请人提供）：胱氨酸病和威尔逊病（WD）的早期诊断至关重要，因为存在有效的治疗方法。不幸的是，大多数患有胱氨酸病或WD的患者是在出现严重并发症后被诊断出来的;特别是肾、肝和脑损伤。对于这两种疾病都没有成本效益高的筛查方法。许多先天性疾病如原发性免疫缺陷病（PIDDs）、胱氨酸病和WD是由导致蛋白质水平缺失或显著降低的突变引起的;因此，位于细胞内（细胞质或跨膜）的蛋白质生物标志物在先天性疾病的诊断/筛查中具有巨大的潜力。我们认为，LC-MRM-MS为基础的方法是有意义的，作为一种快速，廉价的方法，同时筛选各种先天性疾病。作为概念验证，我们已经证明了特征肽的LC-MRM-MS分析可以使用白色血细胞（WBC）鉴定缺乏三种危及生命的PIDD（SCID、WAS和XLA）的特异性蛋白标志物的患者。然而，灵敏度受到样品复杂性的限制，特别是对于直接从DBS检测/定量而不富集的这些低丰度蛋白质。广泛的研究表明，通过肽免疫亲和富集偶联MRM（免疫MRM），可以提高灵敏度的极限，其实现了超过1000倍的富集目标肽。我们的目标是开发和验证一种特异性和定量检测方法，该方法将使用少量血液（包括DBS）同时识别多种遗传疾病。本申请的目的是开发一种基于LC-MRM-MS的方法来量化婴儿血液中的一组生物标志物，以促进两种先天性疾病胱氨酸病和WD的早期检测和诊断。我们的具体目标是：一曰：鉴定胱氨酸蛋白酶（CTNS）、景天庚酮激酶（SHPK）和ATP 7 B的蛋白型肽;胱氨酸蛋白酶增多症和威尔逊病的标志蛋白。我们假设，我们可以定量低丰度蛋白质的LC-MRM-MS方法的基础上，我们的初步数据。我们将首先通过检查人类细胞系来测试我们的假设，以选择这两种疾病的蛋白质型特征肽，并充分优化LC-MRM-MS条件。2.通过将MRM检测与肽免疫亲和富集相结合，提高MRM检测对两种先天性疾病的灵敏度。通过肽免疫亲和富集，我们假设可以同时从少量血液和最终血斑中定量特征肽，从而消除分离白色血细胞的需要。我们将通过生成响应曲线检查每个检测试剂盒的性能指标来检验我们的假设。3.评价多重免疫MRM方法正确识别胱氨酸病或Wilson病患者的能力。我们假设，免疫MRM方法将能够正确地识别患者与胱氨酸或威尔逊病的目标蛋白质是不存在或显着减少。我们将通过回顾性分析临床样本来验证我们的假设。

DESCRIPTION (provided by applicant): The early diagnosis of cystinosis and Wilson disease (WD) is critical, because effective treatments exist. Unfortunately, most patients with cystinosis or WD are diagnosed after developing significant complications; in particular, kidney, liver and brain damage. There are no cost-effective screening methods available for either disorder. Many congenital disorders such as Primary Immunodeficiency diseases (PIDDs), cystinosis, and WD are caused by mutations that result in absent or significantly diminished levels of proteins; thus, protein biomarkers localized within the cells (either cytoplasm or transmembrane) have enormous potential in the diagnosis/screening of congenital disorders. We believe that LC-MRM-MS based approach makes sense as a rapid, inexpensive approach to simultaneously screen for a variety of congenital disorders. As proof-of- concept, we have demonstrated that LC-MRM-MS analysis of signature peptides can identify patients lacking specific protein markers of three life-threatening PIDDs: SCID, WAS, and XLA using White Blood Cells (WBC). However, sensitivity was constrained by sample complexity, especially for these low abundance proteins to be detected/quantified directly from DBS without enrichment. Extensive studies have showed that the limit of sensitivity can be improved by peptide immunoaffinity enrichment coupled to MRM (immuno-MRM), which achieves more than a 1000-fold enrichment for target peptides. Our goal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple genetic conditions using a small volume of blood including DBS. The objective of this application is to develop a LC-MRM-MS based approach to quantify a panel of biomarkers in infant blood to facilitate the early detection and diagnosis of the two congenital disorders, cystinosis and WD. Our specific aims are to: 1: Identify proteotypic peptides for Cystinosin (CTNS), Sedoheptulokinase (SHPK) and ATP7B; marker proteins for Cystinosis and Wilson diseases. We hypothesize that we can quantify low abundance proteins by LC-MRM-MS method based on our preliminary data. We will test our hypothesis first by examining human cell lines to select proteotypic signature peptides for these two diseases and fully optimizing LC-MRM-MS conditions. 2. Increase sensitivity of the MRM assay for the two congenital disorders by coupling it with peptide immunoaffinity enrichment. With peptide immunoaffinity enrichment, we hypothesize that signature peptides can be quantified simultaneously from a small volume of blood and ultimately blood spots, eliminating the need for isolation of white blood cells. We will test our hypothesis by examining the performance metrics for each assay by generating response curves. 3. Evaluate the ability of multiplex immuno-MRM approach to correctly identify patients with cystinosis or Wilson disease. We hypothesize that the immuno-MRM method will be able to correctly identify patients with cystinosis or Wilson disease in which the target proteins are absent or significantly reduced. We will test our hypothesis by analyzing clinical samples retrospectively.