25. Deuterated buffers must be set to pD (not pH). There is a
simple correction factor compensating the difference of measuring deuteron concentration using normal pH electrodes
pD ¼ pH read + 0.41 [42]. Alternatively, pH reading (clearly
marked as such) can be reported instead of the pD value.
26. Samples should be analyzed as soon as possible, e.g., within a
few days. Do not store them for more than 1 week, even at very
low temperatures.
27. Some proteins are not stable enough to allow the preparation
of a fully deuterated control. For the majority of comparative
HDX-MS experiments, its inclusion is anyway not crucial, but
it can offer valuable insight into the back-exchange levels and
provides good quality control of the system used.
28. Tune the mass spectrometer properly to avoid hydrogen scrambling in the ion source [43]. That mainly means lower the
desolvation temperature and possible ion activation during
ion transfer. These low-scrambling parameters may, however,
lead to decrease in signal intensity.
29. It is best not to analyze the samples in any ordered manner,
e.g., sequentially by the conditions or replicates. If the analysis
is stopped or interrupted, recondition the system by running
two or three blanks or a standard protein and two blanks.
Cleaning procedures should be included, and potential carryover checked during the analysis. The biggest source of carryover is the protease column, which can, however, be washed
during each run [40]. Try to keep the system running continuously so that maximal LC reproducibility is maintained.
30. DeutEx is not yet publicly available but will be offered at our
website: http://peterslab.org/downloads.php under HDX
tools section.
31. It might be beneficial to search for all detected charge states
(m/z values) for each peptide, thus not relying only on those
fragmented during the LC-MS/MS run and identified during
the subsequent search. This will yield more data points which
can be either merged and statistically processed or used separately as redundant information supporting the data validity.
32. DeutEx will accept any text form of the input and has automated detection of the content in the columns. If that fails, the
user can correct it and assign the column headers manually.
33. For better clarity, a short video demonstrating the whole workflow is available at http://peterslab.org/downloads/SW/
DeutEx.mp4.
34. In Data Analysis, which is delivered together with MS instruments from Bruker Daltonics, this procedure can be partially
automated using a Visual Basic macro. This macro can be
obtained from the authors upon request.
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simple correction factor compensating the difference of measuring deuteron concentration using normal pH electrodes
pD ¼ pH read + 0.41 [42]. Alternatively, pH reading (clearly
marked as such) can be reported instead of the pD value.
26. Samples should be analyzed as soon as possible, e.g., within a
few days. Do not store them for more than 1 week, even at very
low temperatures.
27. Some proteins are not stable enough to allow the preparation
of a fully deuterated control. For the majority of comparative
HDX-MS experiments, its inclusion is anyway not crucial, but
it can offer valuable insight into the back-exchange levels and
provides good quality control of the system used.
28. Tune the mass spectrometer properly to avoid hydrogen scrambling in the ion source [43]. That mainly means lower the
desolvation temperature and possible ion activation during
ion transfer. These low-scrambling parameters may, however,
lead to decrease in signal intensity.
29. It is best not to analyze the samples in any ordered manner,
e.g., sequentially by the conditions or replicates. If the analysis
is stopped or interrupted, recondition the system by running
two or three blanks or a standard protein and two blanks.
Cleaning procedures should be included, and potential carryover checked during the analysis. The biggest source of carryover is the protease column, which can, however, be washed
during each run [40]. Try to keep the system running continuously so that maximal LC reproducibility is maintained.
30. DeutEx is not yet publicly available but will be offered at our
website: http://peterslab.org/downloads.php under HDX
tools section.
31. It might be beneficial to search for all detected charge states
(m/z values) for each peptide, thus not relying only on those
fragmented during the LC-MS/MS run and identified during
the subsequent search. This will yield more data points which
can be either merged and statistically processed or used separately as redundant information supporting the data validity.
32. DeutEx will accept any text form of the input and has automated detection of the content in the columns. If that fails, the
user can correct it and assign the column headers manually.
33. For better clarity, a short video demonstrating the whole workflow is available at http://peterslab.org/downloads/SW/
DeutEx.mp4.
34. In Data Analysis, which is delivered together with MS instruments from Bruker Daltonics, this procedure can be partially
automated using a Visual Basic macro. This macro can be
obtained from the authors upon request.
216
Ruzena Filandrova et al.