MULTIPLEX ANALYSIS OF INBORN ERRORS OF METABOLISM

先天性代谢缺陷的多重分析

• 批准号: 2893793
• 负责人: Michael H Gelb
• 金额: $ 15.01万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2893793
• 关键词: diagnosis design /evaluation; electrospray ionization mass spectrometry; enzyme deficiency; inborn metabolism disorder; technology /technique development

The majority of recognized genetic diseases are caused by enzyme deficiencies. The advent of molecular biology has allowed a more detailed explanation of how genetic alterations influence protein function, but in most cases DNA analysis will remain a second tier approach to the clinical confirmation of suspected disorders. Enzyme analysis of an appropriate tissue will remain the preferred standard as a measurement of protein function for diagnosis. There are numerous assays of enzyme functions in use, many are tedious, and a variety of different analytical techniques are used to assay the catalytic reactions. The focus of the current proposal is to develop a generally-useful, accurate, and sensitive enzyme assay based on electrospray mass spectrometry. The strategy is to quantify enzymatic reaction velocities by observing the change in mass that the substrate undergoes during the enzymatic reaction. Substrates conjugated to a molecular handle, such as biotin, will be used so that the enzymatic product can be easily purified for mass spectrometry from complex biological fluids by capture with a handle-specific receptor such as streptavidin. With this method, it will be possible to analyze several enzymatic reactions in a single reaction mixture using a single injection into a mass spectrometer (multiple analysis). Electrospray ionization mass spectrometry is an extremely sensitive analytical technique (sub-picomole detection is routinely achieved), and can thus be applied in the situation where only small amounts of biological sample are available. This novel approach has been successfully tested by quantitatively assaying beta-galactosidase in cells from a healthy patient and from an individual lacking this enzyme. The proposed work will be directed at developing novel analyses for the four enzymes that define the Sanfilippo syndrome, two lipid hydrolyzing enzymes that define Niemann-Pick (types A and B) and Krabbe diseases, and two enzymes in the tyrosine breakdown pathway that define tyrosinemia. The development of enzyme assays for several diverse enzymes will allow for multiple enzyme analyses from a single reaction to correctly identify a deficient enzyme that is responsible for similar-appearing genetic disorders and with sensitivity that equals or exceeds those of current methods.

大多数公认的遗传病是由酶缺乏引起的。分子生物学的出现使人们能够更详细地解释基因变化是如何影响蛋白质功能的，但在大多数情况下，DNA分析仍将是临床确认可疑疾病的第二级方法。适当组织的酶分析仍将是用于诊断的蛋白质功能测量的首选标准。在使用中有许多酶功能的分析方法，许多都很繁琐，而且各种不同的分析技术被用来分析催化反应。本方案的重点是建立一种通用、准确、灵敏的基于电喷雾质谱仪的酶分析方法。该策略是通过观察底物在酶反应过程中所经历的质量变化来量化酶反应速度。将使用连接到分子手柄上的底物，如生物素，以便通过使用手柄特定受体(如链霉亲和素)捕获，从复杂的生物液中轻松提纯酶产物用于质谱分析。使用这种方法，可以使用一次进样到质谱仪(多重分析)来分析单一反应混合物中的几个酶反应。电喷雾电离质谱仪是一种非常灵敏的分析技术(通常实现亚皮摩尔检测)，因此可以应用于只有少量生物样本可用的情况。这一新的方法已经通过定量检测来自健康患者和缺乏这种酶的个体的细胞中的β-半乳糖苷酶而成功地进行了测试。拟议的工作将针对定义Sanfilippo综合征的四种酶、定义Niemann-Pick(A型和B型)和Krabbe病的两种脂水解酶以及定义酪氨酸血症的酪氨酸分解途径中的两种酶开发新的分析。对几种不同酶的酶分析的发展将允许从单一反应中进行多种酶分析，以正确地识别导致类似遗传疾病的缺陷酶，并具有等于或超过目前方法的灵敏度。