Multiplex Analysis of Inborn Errors of Metabolism

先天性代谢缺陷的多重分析

• 批准号: 6803043
• 负责人: FRANTISEK TURECEK
• 金额: $ 23.69万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6803043
• 关键词: Multiplex; Analysis; Inborn; Errors; Metabolism

DESCRIPTION (provided by applicant): Enzyme deficiencies are the major cause of genetic diseases. In spite of recent advances in nucleic acid screening technologies that elucidate correlations between genetic alterations, protein expression, and function, it is not yet practical to diagnose individual patients by sequencing their full-length genes. Enzyme analysis of tissue or blood cells remains the preferred standard for measurements of protein function to obtain confirmation of suspected disorders. The main focus of the current proposal is to develop several rapid, generally-useful, accurate, and sensitive enzyme assays based on electrospray ionization mass spectrometry as a single instrumental platform. The strategy is to quantify enzymatic reaction velocities in cultured cell lysates by observing mass changes resulting from an enzyme action on a synthetic substrate-conjugate. Biotin is used as a molecular handle in substrate-conjugates, that allows facile and selective separation of enzymatic products from complex biological mixtures by reversible capture with immobilized streptavidin or monomeric avidin, followed by release for mass spectrometric analysis. Quantitation is achieved by using biotinylated internal standards that are chemically identical to the products of enzymatic reactions, but are distinguished by different molecular mass due to the presence of stable heavy isotopes. Previous results for lysosomal storage diseases showed that this approach allows detection and quantitation of enzymatic products in as little as 2500 cells, makes it possible to analyze two or more enzymatic reactions in a single reaction mixture, and the analytical procedure is readily automated. The proposed work is aimed at developing assays for the enzymes phosphomannomutase, phosphomannoisomerase, dolichol-P mannose synthase, and GlcNAc transferase II to achieve specific diagnoses of the various forms of congenital deficiencies of glycosylation. Another complex group of disorders that will be targeted are the porphyrias that often present perplexing clinical symptoms. The results from the new assays and those developed previously for lysosomal storage diseases will be transferred from the research laboratory to clinical practice. A new technology for the detection of low-level proteins, called Visible Isotope Coded Affinity Tags (VICAT), will be developed to quantify the levels of the structural protein dystrophin, whose deficiency causes Duchenne and Becker muscular dystrophies. In addition to this disease-related application, the VICAT technology will be applicable as a general method for sensitive, specific, and absolute quantitation of cellular proteins.

描述（由申请人提供）： 酶缺乏是遗传疾病的主要原因。尽管核酸筛查技术最近取得了进展，阐明了遗传变异、蛋白质表达和功能之间的相关性，但通过对个体患者的全长基因进行测序来诊断个体患者还不切实际。组织或血细胞的酶分析仍然是测量蛋白质功能以确认疑似疾病的首选标准。 目前的建议的主要重点是开发几种快速，普遍有用的，准确的，和敏感的酶测定的基础上，电喷雾电离质谱作为一个单一的仪器平台。该策略是通过观察合成底物-缀合物上的酶作用引起的质量变化来量化培养的细胞裂解物中的酶促反应速度。生物素被用作底物缀合物中的分子手柄，其允许通过用固定化链霉亲和素或单体亲和素可逆捕获来从复杂的生物混合物中容易和选择性地分离酶促产物，随后释放用于质谱分析。通过使用生物素化内标物实现定量，生物素化内标物与酶促反应产物在化学上相同，但由于存在稳定的重同位素而具有不同的分子量。先前对溶酶体贮积病的结果表明，这种方法允许在少至2500个细胞中检测和定量酶产物，使得可以在单个反应混合物中分析两个或更多个酶反应，并且分析程序易于自动化。 拟议的工作是旨在开发酶磷酸甘露变位酶，磷酸甘露异构酶，dolichol-P甘露糖合成酶，和GlcNAc转移酶II的测定，以实现特定的诊断各种形式的先天性糖基化缺陷。另一组复杂的疾病，将有针对性的卟啉症，往往提出令人困惑的临床症状。来自新测定法和先前开发的用于溶酶体贮积病的测定法的结果将从研究实验室转移到临床实践中。将开发一种用于检测低水平蛋白质的新技术，称为可见同位素编码亲和标签（VICAT），以量化结构蛋白肌营养不良蛋白的水平，其缺陷导致杜氏和贝克尔肌营养不良症。除了这种疾病相关应用外，VICAT技术还可用作细胞蛋白质灵敏、特异和绝对定量的通用方法。