Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
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DOI：
10.1016/j.ab.2014.04.006
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发表时间：
2014-07-01
影响因子：
2.9
通讯作者：
Guianvarc'h, Dominique
中科院分区：
文献类型：
--
作者：
Guitot, Karine;Scarabelli, Silvia;Guianvarc'h, Dominique
组蛋白赖氨酸甲基转移酶（HKMT）是在表观遗传调控中起重要作用的酶。因此，鉴定特异性靶向这些酶的抑制剂代表了开发新的抗肿瘤治疗剂的挑战。已经有几种测量HKMT活性的方法。它们中的大多数通过放射性标记或抗体识别产物或偶联酶测定来间接测量酶促反应。质谱法（MS）代表了一种有趣的替代方法，因为它允许直接检测和定量酶反应，并可用于确定动力学和筛选小分子作为潜在的抑制剂。质谱法在HKMT研究中的应用尚未得到充分探索。在这里，我们描述了一个简单可靠的无标记MALDI-TOF MS为基础的检测和定量的肽甲基化，使用SET 7/9作为模型酶的发展。重要的是，在质谱定量分析中经常需要使用昂贵的内标物，在该测定中是不必要的。该MS测定允许我们确定酶动力学参数以及该酶的已知抑制剂的IC 50。此外，与基于抗体的免疫吸附测定法的比较研究表明，MS测定法更可靠，适用于抑制剂的筛选。(C)2014爱思唯尔公司All rights reserved.
Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. (C) 2014 Elsevier Inc. All rights reserved.