4 Notes
1. The influence of pH and temperature on the intrinsic chemical
exchange rate must be considered here. If two or more states of
a protein are followed at different pH values or temperatures,
appropriate correction must be applied. Temperature factor can
be calculated from the Arrhenius equation (e.g., comparison of
exchange at 10
C and 37
C will be corrected by a factor of
13.88). The effect of pH is compensated by 10ΔpH value (e.g.,
exchange at pH 7 runs 100 times faster than exchange at
pH 5) [3].
2. Total amount depends on protein size, the LC system used,
and the sensitivity of the mass spectrometer. However, tens
(small protein) to hundreds (large protein) of micrograms are
enough for the comparison of two states and performing triplicate analysis.
3. Check the oligonucleotides for possible secondary structure
formation which may affect their binding properties. When
preparing/synthesizing or ordering the oligonucleotides, use
HPLC purification as the last step to obtain desalted DNA
samples.
4. In case where water/ice mixture is used, the valve body should
be properly sealed and isolated from the water (parafilm and
heat shrink tubing) as it prolongs the lifetime of the valve.
5. A wide range of buffer components or concentrations is tolerated in H/D exchange workflow as long as they can be
removed during desalting on a reversed phase resin that precedes separation and mass spectrometric detection. Nevertheless, the buffer components should be kept at their lowest
concentration where both the free protein and the complex
are still stable.
6. At least ten times K D [18]. If K D is not known, try multiple
concentrations. Keep in mind that the complex will be diluted
during D 2 O labeling. Do not use excess of DNA since high
DNA concentration can lead to sample precipitation upon
quenching.
7. Depending on the melting point temperature of the used
dsDNA, cooling might be needed.
8. Try to keep the volume of samples as low as possible to prevent
diffusion from the wells during sample loading—ideally under
5 μl.
9. The concentration of the gel should be adjusted based on
oligonucleotide length.
212
Ruzena Filandrova et al.
1. The influence of pH and temperature on the intrinsic chemical
exchange rate must be considered here. If two or more states of
a protein are followed at different pH values or temperatures,
appropriate correction must be applied. Temperature factor can
be calculated from the Arrhenius equation (e.g., comparison of
exchange at 10
C and 37
C will be corrected by a factor of
13.88). The effect of pH is compensated by 10ΔpH value (e.g.,
exchange at pH 7 runs 100 times faster than exchange at
pH 5) [3].
2. Total amount depends on protein size, the LC system used,
and the sensitivity of the mass spectrometer. However, tens
(small protein) to hundreds (large protein) of micrograms are
enough for the comparison of two states and performing triplicate analysis.
3. Check the oligonucleotides for possible secondary structure
formation which may affect their binding properties. When
preparing/synthesizing or ordering the oligonucleotides, use
HPLC purification as the last step to obtain desalted DNA
samples.
4. In case where water/ice mixture is used, the valve body should
be properly sealed and isolated from the water (parafilm and
heat shrink tubing) as it prolongs the lifetime of the valve.
5. A wide range of buffer components or concentrations is tolerated in H/D exchange workflow as long as they can be
removed during desalting on a reversed phase resin that precedes separation and mass spectrometric detection. Nevertheless, the buffer components should be kept at their lowest
concentration where both the free protein and the complex
are still stable.
6. At least ten times K D [18]. If K D is not known, try multiple
concentrations. Keep in mind that the complex will be diluted
during D 2 O labeling. Do not use excess of DNA since high
DNA concentration can lead to sample precipitation upon
quenching.
7. Depending on the melting point temperature of the used
dsDNA, cooling might be needed.
8. Try to keep the volume of samples as low as possible to prevent
diffusion from the wells during sample loading—ideally under
5 μl.
9. The concentration of the gel should be adjusted based on
oligonucleotide length.
212
Ruzena Filandrova et al.