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

针对胱氨酸病/威尔逊病的肽免疫亲和富集 LC-MRM-MS 分析

• 批准号: 8710295
• 负责人: Sihoun Hahn
• 金额: $ 23.57万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710295
• 关键词: Affect; Amino Acids; Biological Assay; Biological Markers; Blood; Blood Volume; Brain Injuries; Cells; Child; Clinical; Congenital Disorders; Coupled; Coupling; Cystinosis; Cytoplasm; Data; Diagnosis; Disease; Early Diagnosis; Early Intervention; Goals; Health Care Costs; Hepatolenticular Degeneration; Hereditary Disease; Human Cell Line; Individual; Infant; Kidney; Kidney Failure; Lead; Leukocytes; Life; Liquid Chromatography; Liver Cirrhosis; Mass Spectrum Analysis; Measurement; Measures; Methodology; Methods; Metric; Morbidity - disease rate; Neonatal Screening; Newborn Infant; Organ; Outcome; Paper; Patient Care; Patients; Peptides; Performance; Physicians; Post-Translational Protein Processing; Procedures; Proteins; Publishing; Quality of life; Reproducibility; Research; Sampling; Serum; Severe Combined Immunodeficiency; Spottings; Testing; Wilson disease protein; Wiskott-Aldrich Syndrome; X-Linked Agammaglobulinemia; acylcarnitine; base; congenital immunodeficiency; cost effective; disability; disease diagnosis; disease-causing mutation; effective therapy; high throughput screening; improved; liver injury; multiple reaction monitoring; prevent; public health relevance; rapid diagnosis; research study; response; screening; small molecule; tandem mass spectrometry

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.

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，大多数胱氨酸病或WD患者在出现明显并发症后才被诊断出来；尤其是肾、肝和脑损伤。对于这两种疾病，目前都没有成本效益高的筛查方法。许多先天性疾病，如原发性免疫缺陷疾病（PIDDs）、胱氨酸病和WD是由突变引起的，这些突变导致蛋白质水平缺失或显著降低；因此，定位于细胞内（无论是细胞质还是跨膜）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。我们相信基于LC-MRM-MS的方法是一种快速、廉价的方法，可以同时筛查各种先天性疾病。作为概念验证，我们已经证明了LC-MRM-MS分析的特征肽可以识别缺乏三种危及生命的pidd: SCID， WAS和XLA的特定蛋白质标记的患者。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/定量而不富集的低丰度蛋白质。大量研究表明，通过与MRM （immune -MRM）偶联的多肽免疫亲和富集，可以提高敏感性极限，对目标肽的富集可达到1000倍以上。我们的目标是开发并验证一种特定的定量分析方法，该方法将使用包括DBS在内的少量血液同时识别多种遗传疾病。本应用程序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的早期发现和诊断。我们的具体目标是：1 .鉴定胱氨酸蛋白（CTNS）， Sedoheptulokinase （SHPK）和ATP7B的蛋白型肽；胱氨酸病和威尔逊病的标记蛋白。我们假设基于我们的初步数据，我们可以用LC-MRM-MS方法定量低丰度蛋白质。我们将首先通过检测人类细胞系来测试我们的假设，以选择这两种疾病的蛋白型特征肽，并充分优化LC-MRM-MS条件。2. 通过将其与肽免疫亲和富集相结合，增加MRM测定对两种先天性疾病的敏感性。通过多肽免疫亲和富集，我们假设特征肽可以同时从少量血液和最终血斑中定量，从而消除了分离白细胞的需要。我们将通过生成响应曲线来检查每个分析的性能指标来检验我们的假设。3. 评估多重免疫- 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方法将能够正确识别靶蛋白缺失或显著减少的胱氨酸病或Wilson病患者。我们将通过回顾性分析临床样本来检验我们的假设。