examine changes in protein conformation in disease-linked mutations. Importantly, this provides a tool to explore both direct
binding interactions and long range allosteric conformational
changes induced by a variety of binding partners. The more
dynamic a secondary structure element, the faster the exchange
rate. Exchange rates within proteins can vary over a large timescale,
with disordered regions exchanging within milliseconds to seconds,
and extremely stable secondary structure regions exchanging on
the order of hours to days [1]. The underlying theoretical basis for
how and why amide hydrogens are protected from exchange have
been extensively reviewed, and readers are advised to consult the
following reviews for more detail [1–10].
The general flowpath for carrying out an HDX-MS experiment
begins by incubating the protein of interest with deuterated buffer
for a set period of time. During this time, the mass of the protein
will increase as hydrogens are exchanged for deuterium throughout
the protein. A complete HDX-MS experiment requires the analysis
of multiple different time points of deuterium incorporation in
order to obtain a comprehensive understanding of protein dynamics over time. The exchange reaction is terminated by addition of a
low-temperature acidic quench buffer which dramatically slows the
rate of exchange. From there, the protein is digested into peptides
using an acid functional protease such as pepsin to enable spatial
localization of deuterium exchange. Following digestion, an
MS-based mapping approach is utilized which involves separation
of peptides by reverse-phase chromatography and measurement of
peptide masses using a mass spectrometer. Finally, the deuterium
incorporation of each peptide is quantified by examining the shift in
the average mass.
HDX-MS has become a powerful tool for defining protein–
protein [11, 12], protein–ligand [13], and protein–membrane
interactions [14], as well as mapping the effects of posttranslational
modifications [15]. A variety of stimuli can be examined using
HDX-MS including binding partners, small molecules, ligands,
and membrane surfaces. Experiments carried out using membrane
stimuli have already provided substantial insight into our understanding of fundamental signaling processes that occur at membranes. Numerous studies to date have implemented the use of
HDX-MS to characterize protein–membrane interactions for both
integral membrane proteins [16–22] and peripheral membrane
proteins [8, 11, 12, 14, 15, 23–34]. HDX-MS provides a method
to both probe the conformational changes that occur upon membrane binding and defining membrane binding interfaces.
This chapter will describe the HDX-MS methodology used to
study the protein kinase Akt and its interaction with lipid membranes [15]. A brief introduction to Akt will be required to help put
the HDX-MS results in context. Akt is composed of two domains: a
pleckstrin homology (PH) domain that recognizes and binds to
466
Jordan T. B. Stariha et al.
binding interactions and long range allosteric conformational
changes induced by a variety of binding partners. The more
dynamic a secondary structure element, the faster the exchange
rate. Exchange rates within proteins can vary over a large timescale,
with disordered regions exchanging within milliseconds to seconds,
and extremely stable secondary structure regions exchanging on
the order of hours to days [1]. The underlying theoretical basis for
how and why amide hydrogens are protected from exchange have
been extensively reviewed, and readers are advised to consult the
following reviews for more detail [1–10].
The general flowpath for carrying out an HDX-MS experiment
begins by incubating the protein of interest with deuterated buffer
for a set period of time. During this time, the mass of the protein
will increase as hydrogens are exchanged for deuterium throughout
the protein. A complete HDX-MS experiment requires the analysis
of multiple different time points of deuterium incorporation in
order to obtain a comprehensive understanding of protein dynamics over time. The exchange reaction is terminated by addition of a
low-temperature acidic quench buffer which dramatically slows the
rate of exchange. From there, the protein is digested into peptides
using an acid functional protease such as pepsin to enable spatial
localization of deuterium exchange. Following digestion, an
MS-based mapping approach is utilized which involves separation
of peptides by reverse-phase chromatography and measurement of
peptide masses using a mass spectrometer. Finally, the deuterium
incorporation of each peptide is quantified by examining the shift in
the average mass.
HDX-MS has become a powerful tool for defining protein–
protein [11, 12], protein–ligand [13], and protein–membrane
interactions [14], as well as mapping the effects of posttranslational
modifications [15]. A variety of stimuli can be examined using
HDX-MS including binding partners, small molecules, ligands,
and membrane surfaces. Experiments carried out using membrane
stimuli have already provided substantial insight into our understanding of fundamental signaling processes that occur at membranes. Numerous studies to date have implemented the use of
HDX-MS to characterize protein–membrane interactions for both
integral membrane proteins [16–22] and peripheral membrane
proteins [8, 11, 12, 14, 15, 23–34]. HDX-MS provides a method
to both probe the conformational changes that occur upon membrane binding and defining membrane binding interfaces.
This chapter will describe the HDX-MS methodology used to
study the protein kinase Akt and its interaction with lipid membranes [15]. A brief introduction to Akt will be required to help put
the HDX-MS results in context. Akt is composed of two domains: a
pleckstrin homology (PH) domain that recognizes and binds to
466
Jordan T. B. Stariha et al.