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）：酶缺乏是遗传病的主要原因。尽管核酸筛查技术最近取得了进展，阐明了遗传改变、蛋白质表达和功能之间的相关性，但通过对患者的全长基因进行测序来诊断个体患者尚不切实际。组织或血细胞的酶分析仍然是测量蛋白质功能以获得疑似疾病确认的首选标准。当前提案的主要重点是开发几种基于电喷雾电离质谱作为单一仪器平台的快速、通用、准确和灵敏的酶测定方法。该策略是通过观察酶对合成底物缀合物的作用引起的质量变化来量化培养细胞裂解物中的酶反应速度。生物素用作底物缀合物中的分子手柄，通过固定化链霉亲和素或单体亲和素的可逆捕获，可以轻松、选择性地从复杂的生物混合物中分离酶产物，然后释放用于质谱分析。定量是通过使用生物素化的内标来实现的，这些内标在化学上与酶反应的产物相同，但由于存在稳定的重同位素而以不同的分子量来区分。先前关于溶酶体贮积病的结果表明，这种方法可以在少至 2500 个细胞中检测和定量酶产物，使得可以分析单个反应混合物中的两个或多个酶反应，并且分析程序很容易实现自动化。拟议的工作旨在开发磷酸甘露糖变位酶、磷酸甘露糖异构酶、dolichol-P 甘露糖合成酶和 GlcNAc 转移酶 II 的检测方法，以实现对各种形式的先天性糖基化缺陷的特异性诊断。另一组将针对的复杂疾病是经常出现令人困惑的临床症状的卟啉症。新测定法和之前针对溶酶体贮积病开发的测定法的结果将从研究实验室转移到临床实践。将开发一种检测低水平蛋白质的新技术，称为可见同位素编码亲和标签（VICAT），以量化结构蛋白肌营养不良蛋白的水平，这种蛋白的缺乏会导致杜氏肌营养不良症和贝克尔肌营养不良症。除了与疾病相关的应用外，VICAT 技术还可用作细胞蛋白敏感、特异和绝对定量的通用方法。

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。