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陷引起的。 分子生物学的出现允许对遗传改变如何影响蛋白质功能进行更详细的解释，但是在大多数情况下，DNA分析将仍然是临床证实可疑疾病的第二层方法。 适当组织的酶分析将仍然是首选标准，以测量用于诊断的蛋白质功能。有许多使用酶功能的测定法，许多酶繁琐，并且使用多种不同的分析技术来测定催化反应。 当前建议的重点是基于电喷雾质谱法开发一种普遍使用，准确和敏感的酶测定。该策略是通过观察酶促反应过程中底物经历的质量变化来量化酶促反应速度。 将使用与分子手柄（例如生物素）结合的底物，以便通过用手柄特异性受体（如链霉亲素）捕获酶促产物从复杂的生物流体中轻松纯化用于质谱的质谱。 通过这种方法，可以使用单个注射到质谱仪中分析单个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敏感的分析技术（通常可以实现亚曲杆菌检测），因此可以在只有少量生物样品可用的情况下应用。通过定量测定健康患者的细胞和缺乏这种酶的人的细胞中的β-半乳糖苷酶，通过定量测定β-半乳糖苷酶成功测试了这种新颖的方法。 拟议的工作将致力于为定义Sanfilippo综合征的四种酶开发新的分析，这是定义Niemann-Pick（A和B型）和Krabbe疾病的两种脂质水解酶，以及在酪氨酸崩溃途径中定义了定义酪氨酸的两种酶。 几种不同酶的酶测定的开发将允许从单个反应中进行多种酶分析，以正确识别导致类似出现的遗传疾病的缺陷酶，并且具有等于或超过当前方法的敏感性。