Studying Protein–DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry
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Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein–DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.
Key wordsDNA Hydrogen/deuterium exchange Protein–DNA binding Structural mass spectrometry Transcription factor
Czech Science Foundation projects 16-24309S and 16-20860S are gratefully acknowledged. Additional support was obtained from EU/MEYS projects BioCeV (CZ.1.05/1.1.00/02.0109) and NPU II (LQ1604). R.F. also thanks Charles University Grant Agency (project 1618218) and SVV260427/2019.
- 12.Graham BW, Tao Y, Dodge KL et al (2016) DNA interactions probed by hydrogen-deuterium exchange (HDX) Fourier transform ion cyclotron resonance mass spectrometry confirm external binding sites on the minichromosomal maintenance (MCM) helicase. J Biol Chem 291:12467–12480PubMedPubMedCentralCrossRefGoogle Scholar
- 14.Slavata L, Chmelik J, Kavan D et al (2019) MS-based approaches enable the structural characterization of transcription factor/DNA response element complex. Biomol Ther 9:E535Google Scholar
- 18.Kochert BA, Iacob RE, Wales TE et al (2018) Hydrogen-deuterium exchange mass spectrometry to study protein complexes. In: Methods in molecular biology (Clifton, N.J.). Humana Press, New York, NY, pp 153–171Google Scholar