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A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer’s disease drug target β-secretase
Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2018-09-15 , DOI: 10.1007/s00216-018-1354-6
Markéta Machálková , Jan Schejbal , Zdeněk Glatz , Jan Preisler

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer’s disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method’s applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research.

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中文翻译:

一种基于无标记MALDI TOF MS的方法,用于研究阿尔茨海默氏病药物靶β-分泌酶的动力学和抑制剂筛选

基质辅助激光解吸/电离飞行时间质谱(MALDI TOF MS)是一种行之有效的方法,具有一套独特的质量,包括灵敏度,微小的样品消耗和无标签的检测,所有这些都是非常需要的在酶测定中。另一方面,MALDI TOF MS的应用通常受到反应混合物中高浓度的与MS不相容的化合物(例如盐或有机溶剂)的限制。在这里,我们介绍β-分泌酶(BACE1)的动力学和抑制研究,β-分泌酶是阿尔茨海默氏病进展的关键酶。实现了酶分析与MALDI TOF MS的兼容性,既提供了复杂的实验方案,包括旨在进行严格动力学研究的脱盐步骤,又提供了为高通量抑制剂筛选而设计的简单混合测量方案。与荧光或比色法相比,MALDI TOF MS代表了一种灵敏,快速且无标记的技术,只需最少的样品制备。与通常在药物发现过程中使用的其他基于MS的方法学方法(例如直接注射MS或MS耦合的液相色谱法或毛细管电泳)相反,MALDI TOF MS可以进行直接分析,是高度适合高通量筛选的方法。所获得的动力学和抑制参数与文献以及我们以前的研究数据之间的高度相关性极大地支持了该方法的适用性。与通常在药物发现过程中使用的其他基于MS的方法学方法(例如直接注射MS或MS耦合的液相色谱法或毛细管电泳)相反,MALDI TOF MS可以进行直接分析,是高度适合高通量筛选的方法。所获得的动力学和抑制参数与文献数据以及我们先前研究的数据之间的高度相关性,极大地支持了该方法的适用性。与通常在药物发现过程中使用的其他基于MS的方法学方法(例如直接注射MS或MS耦合的液相色谱法或毛细管电泳)相反,MALDI TOF MS可以进行直接分析,是高度适合高通量筛选的方法。所获得的动力学和抑制参数与文献以及我们以前的研究数据之间的高度相关性极大地支持了该方法的适用性。

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更新日期:2018-09-15
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