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Example scenario: MMP2

Make DST
  1. On the "File" menu: select "New Project".

  2. Enter Project Name as "MMP2" and click on "Next >".

  3. Select "mmp2-trypsinfilter.tbt" from the list of theoretical data files and click on "Next >"

  4. Select "62-unmod-tryp-demo.edt" and "72-unmod-tryp-demo.edt" from the list of experimental data files and click on "Next >".

  5. Select "50 ppm" as accuracy value at the bottom of the window and click on "Finish".

Sorting the Project according to amino acid sequence
  1. 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

  2. Go back to default sorting by clicking on the empty title cell of column 1 (above the row numbering in the leftmost column).

  3. Check out what happens if you sort by clicking at the title cell of the "End" column one time or two times.

  4. 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
  1. 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.

  2. Accept the default settings in the SequenceSuggester window, and click "Suggest sequences".

  3. 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.

  4. 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).

  5. 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
  1. 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.

  2. Right-click on one of red balls. Check out the three alternatives you get.

  3. 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).

  4. Try coloring the residues from 31-40 green.

  5. Try coloring C-am modifications yellow.

  6. Try coloring all residues containing Serin (S).

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Download MassSorter v3.1

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Example Scenario: MMP2

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