Example scenario: MMP2
On the "File" menu: select "New Project".
Enter Project Name as "MMP2" and click on "Next >".
Select "mmp2-trypsinfilter.tbt" from the list of theoretical data files
and click on "Next >"
Select "62-unmod-tryp-demo.edt" and "72-unmod-tryp-demo.edt"
from the list of experimental data files and click on "Next >".
Select "50 ppm" as accuracy value at
the bottom of the window and click on "Finish".
Sorting the Project according to amino acid sequence
The default sorting of the Project is according to the m/z values.
The Project may also be sorted according to the amino acid sequence of
the protein. Click at the title cell of the "Start" column. The spread sheet
is then sorted according to the start position in the amino acid
sequence. It is easy to see that the peaks N-terminal to position
115 is lacking in the 62 kD form, but not in the 72 kD form
Go back to default sorting by clicking on the empty title cell
of column 1 (above the row numbering in the leftmost column).
Check out what happens if you sort by clicking at the title
cell of the "End" column one time or two times.
Go back to default sorting by clicking on the empty title
cell of column 1. For other types of analyses (like using
SequenceSuggester or UniModSearch), the spread sheet must be in the default sorting.
Redefinition of unmatched masses as matched masses
A peak close to 508 is found in both 62 and 72 kD version of MMP.
Right-click on one of the cells with this value (row number one). Select
"Suggest sequence(s)" in the popup menu.
Accept the default settings in the SequenceSuggester window, and
click "Suggest sequences".
A set of sequences then appears. One of the suggestions is the
sequence "FWR", which would fit with tryptic cleavage at either side
of the peptide, but it has not been included as it consists of only
three amino acids. Select the row containing the "FWR" peptide. The
selected sequence will then be highlighted in blue in the protein sequence
at the upper right. Now right-click on the "FWR" line, and click on
"Insert into DST". Answer "yes" to the questions. The 508 peak is
now highlighted in blue in the DST.
Similarly, a common peak at 1392 is unmatched. Again try to use
the "SequenceSuggester" on this peak. The most likely suggestion is
the peptide 555-567 (PK)-PLTSLGLPPDVQR, as the sequence PKP is
sensitive to tryptic cleavage in contrast to all other XKP sequences
(A. Gattiker et al., Proteomics 2, 1435-1444, 2002).
In the DST a peak at 843.4 is present only in the 62 kD form.
Try the "SequenceSuggester" on this peak. One of the suggestions is
the peptide 110-115 YNFFPR. The accuracy is not among the best, but
this is at least partly due to the overlap with the trypsin peak at
842.5. Insert the match into the DST and try to sort the DST according
to the amino acid sequence (click on title cell of "Start" column),
and you immediately see that this fits nicely with the other data.
This is in fact the correct N-terminus of the 62 kD form. Go back to
the default sorting (click on empty title cell in the left-most column).
Viewing the results in a 3D model
If a PDB file is available for your protein, you can look at your
results on a 3D model. On the "Tools" menu, select "Report". Above each
of the protein sequences where the detected peptides are marked in red,
click on "View as 3D model". Find the relevant PDB file (which you have
downloaded) and open it, in this case use "mmp2.pdb", which can be found
in the "DataFiles" folder. After some seconds, the 3D model appears. The
detected peptides are marked in red. Click (and hold) somewhere on the
protein, then move the cursor slowly to turn the molecule.
Right-click on one of red balls. Check out the three alternatives
In the "ProteinViewer", go to select "Color-coding" on the
"Color" menu. In this window you can color i) the detected and
undetected peptides ii) the modifications for which you would like
to check the positions (e.g., is a phosphorylation positioned at
the external surface of the molecule?); iii) any amino acids (e.g.,
are there negatively charged amino acids in a certain area of the
3D structure?); iii) any specific position (e.g., position 110: put
110 into both cells) or peptide (e.g., 110-115).
Try coloring the residues from 31-40 green.
Try coloring C-am modifications yellow.
Try coloring all residues containing Serin (S).
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Example Scenario: MMP2
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