Chapter 22
Probing Protein–Membrane Interactions and Dynamics
Using Hydrogen–Deuterium Exchange Mass Spectrometry
(HDX-MS)
Jordan T. B. Stariha, Reece M. Hoffmann, David J. Hamelin,
and John E. Burke
Abstract
Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to
these processes being accomplished appropriately is the highly controlled recruitment and assembly of
proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein–membrane
interactions can be facilitated by utilizing hydrogen–deuterium exchange mass spectrometry (HDX-MS).
HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone
amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter
discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral
membrane proteins upon association with membrane. Particular reference will be made to the analysis of
the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP 3 )containing membranes to illustrate specific methodological principles.
Key words HDX-MS, Hydrogen–deuterium exchange, Structural proteomics, Mass spectrometry,
Protein–membrane interactions, Lipid signaling, Protein dynamics
1 Introduction
Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a
biophysical technique that probes conformational protein dynamics
through the measurement of the exchange rate of amide hydrogens
with deuterated solvent. The basis of the technique relies on the
fact that amide hydrogens participate in hydrogen bond networks
to form secondary structural elements such as alpha helices and beta
sheets. The exchange rate of amide hydrogens is exquisitely sensitive to the presence and dynamics of these secondary structure
elements. The differences in secondary structure stability can be
used as a probe to examine differences upon binding to ligand,
protein, and membrane-binding partners. This can also be used to
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
465
Probing Protein–Membrane Interactions and Dynamics
Using Hydrogen–Deuterium Exchange Mass Spectrometry
(HDX-MS)
Jordan T. B. Stariha, Reece M. Hoffmann, David J. Hamelin,
and John E. Burke
Abstract
Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to
these processes being accomplished appropriately is the highly controlled recruitment and assembly of
proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein–membrane
interactions can be facilitated by utilizing hydrogen–deuterium exchange mass spectrometry (HDX-MS).
HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone
amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter
discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral
membrane proteins upon association with membrane. Particular reference will be made to the analysis of
the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP 3 )containing membranes to illustrate specific methodological principles.
Key words HDX-MS, Hydrogen–deuterium exchange, Structural proteomics, Mass spectrometry,
Protein–membrane interactions, Lipid signaling, Protein dynamics
1 Introduction
Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a
biophysical technique that probes conformational protein dynamics
through the measurement of the exchange rate of amide hydrogens
with deuterated solvent. The basis of the technique relies on the
fact that amide hydrogens participate in hydrogen bond networks
to form secondary structural elements such as alpha helices and beta
sheets. The exchange rate of amide hydrogens is exquisitely sensitive to the presence and dynamics of these secondary structure
elements. The differences in secondary structure stability can be
used as a probe to examine differences upon binding to ligand,
protein, and membrane-binding partners. This can also be used to
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
465