Screening of protein-ligand interactions under crude conditions by native mass spectrometry
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Screening of protein-ligand interactions under crude conditions by native mass spectrometry Kotaro Takano 1 & Shunsuke Arai 1 & Seiji Sakamoto 2 & Hiroshi Ushijima 1 & Takahisa Ikegami 1 & Kazumi Saikusa 1,3 & Tsuyoshi Konuma 1 & Itaru Hamachi 2 & Satoko Akashi 1 Received: 5 March 2020 / Revised: 1 April 2020 / Accepted: 6 April 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 DHFRNADPH-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 ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in “real” biological systems. Keywords Native mass spectrometry . Cell lysate . Crude conditions . Protein-ligand interactions
Introduction Protein-ligand interactions play crucial roles in various biological events. In addition, small molecules, such as cofactors of proteins, are involved in various processes in living cells. To inhibit the function of pathogenic proteins, a specific Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02649-x) contains supplementary material, which is available to authorized users. * Satoko Akashi [email protected] 1
Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
2
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
3
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8563, Japan
inhibitor binding to the active site of the target is necessary. To determine the biologically significant interactions between a protein and small m
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