7. The basic output from DeutEx is a set of uptake plots that are
displayed either as deuteration percentage or number of
exchanged deuterons as a function of time. The data can also
be exported in simple text form which allows their further
processing in Excel or MSTools (http://peterslab.org/
MSTools/) [38].
3.5.2 Workflow B:
Manual Data Interpretation
1. In any program that is used for viewing the LC-MS data, open
a non-deuterated LC-MS analysis.
2. For each peptide, trace an extracted ion chromatogram based
on its m/z (usually included in the search engine report).
Check also other charge states that are visible and enter this
information into an Excel sheet together with the retention
time limits for further use. Sum scans across the LC peak and
export the spectrum as a simple text file (typically two columns
including m/z values and intensity).
3. Open a partially deuterated LC-MS analysis and locate the
deuterated signal corresponding to the peptide information
gathered in the previous step. Repeat the step of summing up
the scans and exporting the spectra into text files. Repeat
iteratively for all peptides. For such a manual approach, it is
advisable to select a minimal set of peptides (preferably short
ones) covering the entire protein without many overlaps (see
Note 34).
4. Once the data are exported to *.txt files, run mMass program
[39] (see Note 35). Go to File>Open and open a file with
non-deuterated data. The spectrum is not by default labeled,
but it is not necessary at this point. Make sure that the correct
way of peak picking is selected. Go to tab Processing>Deisotoping and set “Label envelope tool” to Envelope Centroid option.
Now locate the selected peak (m/z) and zoom to the mass
range covering the whole isotopic pattern of this peptide. Use
“Label envelope tool” (select from tab Tools). Using Shift +
mouse middle wheel scrolling, set the desired charge state—
black dots are changing distance as you scroll up and down. Fit
it to the desired isotopomer distance and now select the number of dots to cover all visible isotopic peaks from the particular
envelope. This is done by Ctrl + Shift + mouse middle button
scrolling. This tool helps you to localize all isotopes belonging
to the selected peak. Left click and the peak will be labeled.
Average m/z value is written into the spectrum, and the deconvoluted value (average mass) appears in the right panel (Fig. 5).
Write this number down to an Excel file. Eventually, you can
now repeat this step also for other charge states of the same
peptide. One by one, all m/z values of each peptide from all
deuterated samples are processed using this approach (see
Note 36).
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displayed either as deuteration percentage or number of
exchanged deuterons as a function of time. The data can also
be exported in simple text form which allows their further
processing in Excel or MSTools (http://peterslab.org/
MSTools/) [38].
3.5.2 Workflow B:
Manual Data Interpretation
1. In any program that is used for viewing the LC-MS data, open
a non-deuterated LC-MS analysis.
2. For each peptide, trace an extracted ion chromatogram based
on its m/z (usually included in the search engine report).
Check also other charge states that are visible and enter this
information into an Excel sheet together with the retention
time limits for further use. Sum scans across the LC peak and
export the spectrum as a simple text file (typically two columns
including m/z values and intensity).
3. Open a partially deuterated LC-MS analysis and locate the
deuterated signal corresponding to the peptide information
gathered in the previous step. Repeat the step of summing up
the scans and exporting the spectra into text files. Repeat
iteratively for all peptides. For such a manual approach, it is
advisable to select a minimal set of peptides (preferably short
ones) covering the entire protein without many overlaps (see
Note 34).
4. Once the data are exported to *.txt files, run mMass program
[39] (see Note 35). Go to File>Open and open a file with
non-deuterated data. The spectrum is not by default labeled,
but it is not necessary at this point. Make sure that the correct
way of peak picking is selected. Go to tab Processing>Deisotoping and set “Label envelope tool” to Envelope Centroid option.
Now locate the selected peak (m/z) and zoom to the mass
range covering the whole isotopic pattern of this peptide. Use
“Label envelope tool” (select from tab Tools). Using Shift +
mouse middle wheel scrolling, set the desired charge state—
black dots are changing distance as you scroll up and down. Fit
it to the desired isotopomer distance and now select the number of dots to cover all visible isotopic peaks from the particular
envelope. This is done by Ctrl + Shift + mouse middle button
scrolling. This tool helps you to localize all isotopes belonging
to the selected peak. Left click and the peak will be labeled.
Average m/z value is written into the spectrum, and the deconvoluted value (average mass) appears in the right panel (Fig. 5).
Write this number down to an Excel file. Eventually, you can
now repeat this step also for other charge states of the same
peptide. One by one, all m/z values of each peptide from all
deuterated samples are processed using this approach (see
Note 36).
208
Ruzena Filandrova et al.