quench buffer) artificial, nonnative, in-exchange. In contrast,
robotics can set up the mixing of H/D exchange samples
consecutively, so that once the exchange is finished by the
quench solution addition, the sample is directly injected for
analysis. It is also of high importance that some proteins tend
to precipitate during freezing/thawing procedures and here
the use of robotics provides additional advantage.
21. Dilution is often done ten-fold, but higher (20-fold) or lower
(five-fold) ratios are also possible. The key consideration here is
the stability of the complex at a given concentration which is
influenced by the K D of the complex.
22. The amount/volume of the quench solution is given by the
quench ratio. Optimally, ten-fold dilution is used but other
ratios, e.g., 1:1 are also acceptable, if necessary. Quench solution must be acidic and strong enough to override pH of the
labeling buffer and should make it 2.3–2.5, instead. Diluted
solutions of hydrochloric or phosphoric acid, phosphate buffers, or glycine hydrochloride buffers are typical examples.
Quench solution may also contain denaturing agents or other
components necessary for the solubility of the protein or its
analysis. Only components that are not easily removed by
reversed phase desalting and are not compatible with ESI-MS
should be avoided (e.g., detergents). However, even for nonionic detergents, specific workflows allowing their nearly complete removal under HDX-MS conditions have been
described [41].
The tubes used for freezing should be PCR tubes as they
have to be stable at extreme temperature. Normal Eppendorf
microcentrifuge tubes can break or take up some liquid nitrogen, which can lead to their explosion during the subsequent
thawing procedure.
23. Preferably run H/D exchange with several time points spanning the region from seconds to a couple of hours. For longer
incubation times, a control of protein stability should be performed by incubating the protein for the longest selected time
in the normal buffer and then deuterium labeling it for
the shortest selected period (e.g., 20 s). If the deuteration is
the same as for the sample just deuterated for this time window,
the protein is probably stable. If the deuteration profile differs/
is indicative of protein unfolding, shorter time windows should
be sought or the exchange should be done at lower
temperature.
24. Simple setup requires two protein states: protein alone and
protein in complex with its DNA binding partner. However,
number of conditions to be followed can be much larger, e.g.,
with different protein:DNA ratios or different ligands.
HDX-MS of Protein-DNA Complexes
215
robotics can set up the mixing of H/D exchange samples
consecutively, so that once the exchange is finished by the
quench solution addition, the sample is directly injected for
analysis. It is also of high importance that some proteins tend
to precipitate during freezing/thawing procedures and here
the use of robotics provides additional advantage.
21. Dilution is often done ten-fold, but higher (20-fold) or lower
(five-fold) ratios are also possible. The key consideration here is
the stability of the complex at a given concentration which is
influenced by the K D of the complex.
22. The amount/volume of the quench solution is given by the
quench ratio. Optimally, ten-fold dilution is used but other
ratios, e.g., 1:1 are also acceptable, if necessary. Quench solution must be acidic and strong enough to override pH of the
labeling buffer and should make it 2.3–2.5, instead. Diluted
solutions of hydrochloric or phosphoric acid, phosphate buffers, or glycine hydrochloride buffers are typical examples.
Quench solution may also contain denaturing agents or other
components necessary for the solubility of the protein or its
analysis. Only components that are not easily removed by
reversed phase desalting and are not compatible with ESI-MS
should be avoided (e.g., detergents). However, even for nonionic detergents, specific workflows allowing their nearly complete removal under HDX-MS conditions have been
described [41].
The tubes used for freezing should be PCR tubes as they
have to be stable at extreme temperature. Normal Eppendorf
microcentrifuge tubes can break or take up some liquid nitrogen, which can lead to their explosion during the subsequent
thawing procedure.
23. Preferably run H/D exchange with several time points spanning the region from seconds to a couple of hours. For longer
incubation times, a control of protein stability should be performed by incubating the protein for the longest selected time
in the normal buffer and then deuterium labeling it for
the shortest selected period (e.g., 20 s). If the deuteration is
the same as for the sample just deuterated for this time window,
the protein is probably stable. If the deuteration profile differs/
is indicative of protein unfolding, shorter time windows should
be sought or the exchange should be done at lower
temperature.
24. Simple setup requires two protein states: protein alone and
protein in complex with its DNA binding partner. However,
number of conditions to be followed can be much larger, e.g.,
with different protein:DNA ratios or different ligands.
HDX-MS of Protein-DNA Complexes
215