MULTIPLEX ANALYSIS OF INBORN ERRORS OF METABOLISM

先天性代谢缺陷的多重分析

• 批准号: 6387033
• 负责人: Michael H Gelb
• 金额: $ 15.91万
• 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 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387033
• 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.

大多数公认的遗传疾病是由酶缺乏引起的。分子生物学的出现使得对基因改变如何影响蛋白质功能有了更详细的解释，但在大多数情况下，DNA分析仍将是临床确认疑似疾病的第二级方法。适当组织的酶分析仍然是诊断蛋白质功能测量的首选标准。酶的功能测定方法很多，但很多都很繁琐，催化反应的分析技术也多种多样。当前建议的重点是开发一种基于电喷雾质谱的通用、准确和敏感的酶分析方法。该策略是通过观察底物在酶促反应过程中所经历的质量变化来量化酶促反应速度。结合到分子柄的底物，如生物素，将被使用，这样酶产物可以很容易地从复杂的生物流体中通过捕获特定的柄受体（如链霉亲和素）进行质谱纯化。使用这种方法，可以在质谱仪中使用单次注射来分析单一反应混合物中的几种酶促反应（多重分析）。电喷雾电离质谱法是一种非常灵敏的分析技术（亚皮摩尔检测是常规实现），因此可以在只有少量生物样品可用的情况下应用。这种新方法已经通过定量分析来自健康患者和缺乏这种酶的个体的细胞中的-半乳糖苷酶成功地进行了测试。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病的两种脂质水解酶，以及定义酪氨酸血症的酪氨酸分解途径中的两种酶，开发新的分析方法。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，以正确识别导致类似遗传疾病的缺陷酶，其灵敏度等于或超过当前方法。