Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 412, Issue 17, Pages 4037-4043Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-020-02649-x
Keywords
Native mass spectrometry; Cell lysate; Crude conditions; Protein-ligand interactions
Funding
- JSPS KAKENHI [JP18H04561, JP17K07313, JP19H05774, JP19K16091]
- Platform Project for Supporting Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED) [JP19am0101076]
- Nakatani Foundation
- JKA Foundation
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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 <= 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. Graphical abstract
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