Multiplex Analysis of Inborn Errors of Metabolism

先天性代谢缺陷的多重分析

基本信息

批准号：
6929091
负责人：
FRANTISEK TURECEK
金额：
$ 23.18万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
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/6929091
关键词：
Multiplex Analysis Inborn Errors Metabolism

项目摘要

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）：酶缺陷是遗传疾病的主要原因。尽管最近在核酸筛选技术方面取得了进步，这些技术阐明了遗传改变，蛋白质表达和功能之间的相关性，但通过对其全长基因进行测序来诊断单个患者尚不实际。组织或血细胞的酶分析仍然是测量蛋白质功能的首选标准，以确认可疑疾病。当前建议的主要重点是基于电喷雾电离质谱法作为单个仪器平台，开发出几种快速，普遍使用，准确和敏感的酶测定。该策略是通过观察酶作用对合成底物偶联物引起的质量变化来量化培养细胞裂解物中的酶促反应速度。生物素用作底物 - 偶联物中的分子手柄，可以通过固定的链霉亲素或单体抗生物素蛋白可逆地捕获酶促的酶产物从复杂的生物混合物中易于分离，然后释放进行质谱分析。定量是通过使用与酶促反应产物相同的生物素化的内部标准标准来实现的，但由于存在稳定的重量同位素而以不同的分子质量区分。溶酶体储存疾病的先前结果表明，这种方法允许在低至2500个细胞中检测和定量酶促产物，这使得可以在单个反应混合物中分析两种或多种酶促反应，并且很容易自动自动化分析程序。拟议的工作旨在开发用于磷酸磷酸酶，磷诺异分酶，Dolichol-P甘露糖合酶和GlcNAC转移酶II的测定法，以实现各种形式的糖基化先天性缺陷的特定诊断。另一个将要针对的疾病是卟啉症，通常会出现困惑的临床症状。新测定的结果以及先前针对溶酶体储存疾病开发的结果将从研究实验室转移到临床实践。将开发一种用于检测低水平蛋白的新技术，称为可见同位素编码的亲和力标签（VICAT），以量化结构性蛋白质障碍蛋白的水平，其缺乏会导致Duchenne和Becker肌肉发育不全。除了这种与疾病相关的应用外，VICAT技术还将作为敏感，特异性和绝对定量细胞蛋白的一般方法。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib.

磷酸甘露糖变位酶和磷酸甘露糖异构酶的亲和捕获和洗脱/电喷雾电离质谱测定，用于糖基化类型 Ia 和 Ib 先天性疾病的多重分析。

DOI：
10.1021/ac0205053
发表时间：
2003
期刊：
Analytical chemistry
影响因子：
7.4
作者：
Li,Yijun;Ogata,Yuko;Freeze,HudsonH;Scott,CRonald;Turecek,Frantisek;Gelb,MichaelH
通讯作者：
Gelb,MichaelH

Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry.

通过电喷雾电离质谱法定量细胞酸性鞘磷脂酶和半乳脑苷 β-半乳糖苷酶活性。