10. If possible, cool the electrophoresis apparatus during the run
since the complex or DNA duplex might not be stable at higher
temperatures.
11. It should be considered that the fragmentation of a particular
peptide does not always lead to sufficient number of fragments.
Therefore, the combination of different digestion protocols
and dissociation techniques usually represents the best
solution.
12. Fragments covering the same region of protein and differing in
just few amino acids from the N- or C-terminus should result
in fairly similar H/D uptake plots. Hence, the slope, level of
deuteration, or eventually the differences between the individual studied states should not be vastly different. If they are, this
might be an indication of a possible data processing error. On
the other hand, if there are multiple individual peptides spanning one protein region and all show highly similar trend, the
confidence in the observed changes is significantly increased.
13. Protease column may suffer from autodigestion upon longer
storage. Storing it at conditions that do not affect its activity,
while preventing autodigestion (e.g., citrate buffer, pH 4–5,
4
C), is highly recommended. Also, pre-washing with the
loading solvent before connecting the column in front of the
trap column should be routinely done during an LC system
setup.
14. Specific cleaning solutions containing denaturing agents or
higher concentration of acids can be designed to minimize
carry-over [40]. Good regeneration of the whole system is
likely achieved by combining injection of some other (quality
control) protein together with these washing steps.
15. Quench conditions (quench solution composition, concentration, quench ratio) should be tuned prior to the experiment
using pure buffers. While protein concentration during the
labeling step is given by the requirements on the protein–
DNA complex formation, the final concentration and sample
volume after quenching depend solely on the requirements of
the mass spectrometer and LC setup (injection volume, trap,
and protease column size + flow). For the MS analysis, it is
worth not considering the true detection limits of the instrument, but staying well above them, as the signal intensity after
labeling decreases due to signal spreading to multiple isotopes
upon deuteration. Keeping the same protein concentration for
LC-MS/MS as for the subsequent LC-MS analyses is crucial as
the protein:protease ratio affects the digestion pattern.
16. Sum the volumes of injection loop, protease column, trap
column, and the connecting tubing and compare it to
the selected flow on loading/desalting pump. Make sure that
HDX-MS of Protein-DNA Complexes
213
since the complex or DNA duplex might not be stable at higher
temperatures.
11. It should be considered that the fragmentation of a particular
peptide does not always lead to sufficient number of fragments.
Therefore, the combination of different digestion protocols
and dissociation techniques usually represents the best
solution.
12. Fragments covering the same region of protein and differing in
just few amino acids from the N- or C-terminus should result
in fairly similar H/D uptake plots. Hence, the slope, level of
deuteration, or eventually the differences between the individual studied states should not be vastly different. If they are, this
might be an indication of a possible data processing error. On
the other hand, if there are multiple individual peptides spanning one protein region and all show highly similar trend, the
confidence in the observed changes is significantly increased.
13. Protease column may suffer from autodigestion upon longer
storage. Storing it at conditions that do not affect its activity,
while preventing autodigestion (e.g., citrate buffer, pH 4–5,
4
C), is highly recommended. Also, pre-washing with the
loading solvent before connecting the column in front of the
trap column should be routinely done during an LC system
setup.
14. Specific cleaning solutions containing denaturing agents or
higher concentration of acids can be designed to minimize
carry-over [40]. Good regeneration of the whole system is
likely achieved by combining injection of some other (quality
control) protein together with these washing steps.
15. Quench conditions (quench solution composition, concentration, quench ratio) should be tuned prior to the experiment
using pure buffers. While protein concentration during the
labeling step is given by the requirements on the protein–
DNA complex formation, the final concentration and sample
volume after quenching depend solely on the requirements of
the mass spectrometer and LC setup (injection volume, trap,
and protease column size + flow). For the MS analysis, it is
worth not considering the true detection limits of the instrument, but staying well above them, as the signal intensity after
labeling decreases due to signal spreading to multiple isotopes
upon deuteration. Keeping the same protein concentration for
LC-MS/MS as for the subsequent LC-MS analyses is crucial as
the protein:protease ratio affects the digestion pattern.
16. Sum the volumes of injection loop, protease column, trap
column, and the connecting tubing and compare it to
the selected flow on loading/desalting pump. Make sure that
HDX-MS of Protein-DNA Complexes
213