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。 分子生物学的出现允许更详细地解释遗传改变如何影响蛋白质功能，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层方法。 适当组织的酶分析将仍然是诊断蛋白质功能测量的首选标准。有许多酶功能的测定在使用中，许多是繁琐的，并且各种不同的分析技术用于测定催化反应。 目前的建议的重点是开发一种普遍有用的，准确的，灵敏的酶测定电喷雾质谱法的基础上。该策略是通过观察底物在酶促反应期间经历的质量变化来量化酶促反应速度。 将使用与分子柄（例如生物素）缀合的底物，使得酶产物可以容易地从复杂的生物流体中纯化用于质谱分析，通过用生物素特异性受体（例如链霉抗生物素蛋白）捕获。 使用这种方法，将有可能使用单次进样到质谱仪中来分析单个反应混合物中的几个酶反应（多重分析）。 电喷雾电离质谱是一种非常灵敏的分析技术（常规实现亚皮摩尔检测），因此可以应用于只有少量生物样品可用的情况。这种新的方法已经成功地测试了定量测定β-半乳糖苷酶的细胞从一个健康的病人和一个人缺乏这种酶。 拟议的工作将针对开发新的分析定义的Sanfilippo综合征的四种酶，两种脂质水解酶，定义尼曼-皮克（A型和B）和克拉伯病，和两种酶的酪氨酸分解途径，定义酪氨酸血症。 几种不同酶的酶测定的发展将允许从一个单一的反应，以正确地识别一个缺陷的酶，是负责类似的外观遗传疾病，并具有等于或超过目前的方法的灵敏度的多个酶分析。