High-Throughput MALDI-TOF Mass Spectrometry-Based Deubiquitylating Enzyme Assay for Drug Discovery.
High-Throughput MALDI-TOF Mass Spectrometry-Based Deubiquitylating Enzyme Assay for Drug Discovery.
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DOI：
10.1007/978-1-0716-2803-4_8
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发表时间：
2023-01-01
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影响因子：
--
通讯作者：
Davies, Paul
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文献类型：
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作者：
De Cesare, Virginia;Davies, Paul
泛素化（或泛素化）是 76 个氨基酸多肽（泛素）与靶蛋白的可逆缀合，以调节各种生物过程。去泛素化酶 (DUB) 是一类专门从底物中去除泛素的酶。近年来，DUB 作为多个治疗领域的一类新靶点受到了广泛关注。最近发展的高通量基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）为药物发现筛选提供了新工具。在这里，我们提出了一种基于 MALDI-TOF MS 的 DUB 测定的简便且高通量的分步方案，用于体外筛选 DUB 的活性。在 MALDI-TOF DUB 测定中，我们定量了泛素链体外裂解产生的单泛素的量。所提出的协议利用纳升分配机器人技术和自动化 MALDI-TOF MS 分析来筛选大型且多样化的化合物库。
Ubiquitylation (or ubiquitination) is the reversible conjugation of a 76-amino-acid polypeptide (ubiquitin) to a target protein to modulate various biological processes. Deubiquitylating enzymes (DUBs) are a class of enzymes that specifically remove ubiquitin from a substrate. In recent years DUBs have garnered significant attention as a new class of targets in multiple therapeutic areas. The recent development of high-throughput Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has provided new tools to perform drug discovery screening. Here we present a facile and high-throughput step-by-step protocol of the MALDI-TOF MS-based DUB assay for screening the activity of DUBs in vitro. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains. The presented protocol takes advantage of nanoliter dispensing robotics and automated MALDI-TOF MS analysis to screen large and diverse compound libraries.