Chapter 11
Studying Protein–DNA Interactions by Hydrogen/Deuterium
Exchange Mass Spectrometry
Ruzena Filandrova, Daniel Kavan, Alan Kadek, Petr Novak, and Petr Man
Abstract
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 words DNA, Hydrogen/deuterium exchange, Protein–DNA binding, Structural mass spectrometry, Transcription factor
1 Introduction
Structural proteomics is a rapidly developing field focusing specifically on protein structural dynamics and characterization of the
architecture of protein macromolecular assemblies. Hydrogen/
deuterium exchange (HDX) ranks as one of the most versatile and
prominent methods in this area. Its biggest advantages are not
being limited by protein size and the ability to study proteins in
their truly native-like environment, at virtually any pH, buffer
composition, protein concentration, or temperature (see Note 1).
In a typical setup, an HDX experiment starts by the dilution of a
protein into a deuterated buffer. At selected time points, aliquots
are taken, and the exchange is quenched (nearly stopped) by rapid
acidification to pH 2.5 and by lowering the temperature to 0
C.
Subsequently, each sample is digested by acidic, nonspecific proteases, and deuteration of the generated peptides is measured by
mass spectrometry (MS) [1–3].
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
193
Studying Protein–DNA Interactions by Hydrogen/Deuterium
Exchange Mass Spectrometry
Ruzena Filandrova, Daniel Kavan, Alan Kadek, Petr Novak, and Petr Man
Abstract
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 words DNA, Hydrogen/deuterium exchange, Protein–DNA binding, Structural mass spectrometry, Transcription factor
1 Introduction
Structural proteomics is a rapidly developing field focusing specifically on protein structural dynamics and characterization of the
architecture of protein macromolecular assemblies. Hydrogen/
deuterium exchange (HDX) ranks as one of the most versatile and
prominent methods in this area. Its biggest advantages are not
being limited by protein size and the ability to study proteins in
their truly native-like environment, at virtually any pH, buffer
composition, protein concentration, or temperature (see Note 1).
In a typical setup, an HDX experiment starts by the dilution of a
protein into a deuterated buffer. At selected time points, aliquots
are taken, and the exchange is quenched (nearly stopped) by rapid
acidification to pH 2.5 and by lowering the temperature to 0
C.
Subsequently, each sample is digested by acidic, nonspecific proteases, and deuteration of the generated peptides is measured by
mass spectrometry (MS) [1–3].
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
193