Screening of protein-ligand interactions under crude conditions by native mass spectrometry
Screening of protein-ligand interactions under crude conditions by native mass spectrometry
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DOI：
10.1007/s00216-020-02649-x
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发表时间：
2020-04-23
影响因子：
4.3
通讯作者：
Akashi, Satoko
中科院分区：
文献类型：
--
作者：
Takano, Kotaro;Arai, Shunsuke;Akashi, Satoko
一个方便的分析系统的蛋白质-配体的相互作用，在原油条件下，开发使用天然质谱（MS）。以大肠杆菌（Escherichia coli，E.大肠杆菌）二氢叶酸还原酶（DHFR）用于该研究。首先，用Ni-琼脂糖树脂粗略纯化过表达的具有His标签的DHFR，并在与抑制剂甲氨蝶呤（MTX）孵育或不孵育的情况下进行天然质谱分析。即使仅通过Ni-琼脂糖树脂进行最小限度的清除，也成功观察到了DHFR-烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和DHFR-NADPH-配体复合物的完整离子。通过优化天然MS的粗样品制备程序，例如，避免了用于细胞裂解的超声处理，我们成功地从培养大肠杆菌开始观察到特异性DHFR-NADPH-MTX三元复合物的完整离子。杆菌
A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in