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Now that you know what MassSorter is (and is not), how to start using MassSorter? This tutorial explains the basic features and procedures, and makes you able to understand the essential ideas behind MassSorter.

  1. Before starting to use MassSorter you need the amino acid sequence of the protein you want to study. In this tutorial we will use an example sequence.

  2. Comparing experimental and theoretical data is the main idea in MassSorter. This information is represented in a Project. To create a new Project, select "New Project" from the "File" menu in the main window. A new Project wizard then appears which will guide you through the process of creating a Project. The wizard consists of four steps.

  3. The first step is inserting the Project details. Provide a Project name and description. Only the name is mandatory, but inserting a description is highly recommended. Click on "Next >" to continue.

  4. The second step is providing the theoretical data. Here you have two choices; you can either create a new theoretical data file from scratch using MassSorter's own tool ProteinDigester, or you can select one from the list of the allready created data files. In this tutorial we will we will choose the first option. Click on the "ProteinDigester" button. This will open a protein digester window. Select "Import sequence" from the "File menu", select the text file named "BSA" and click on "Open". Now you can select the parameters for the digestion. In the modifications list select the modifications named C-am, gK, oM and pyrE. If you want to see more information about the modifications right click on the given abbreviation in the first column. Leave the other parameters on the default settings and click on "Digest protein".

  5. The ProteinDigester window then disappears, and the newly created data file is inserted in the list of available data files and selected. To see the contents of the file, right click on the given row and select "Preview Theoretical Data File" from the popup menu. A window appears containing the data about the theoretical digestion. Each line contains information about one peptide: the mass m/z value, start- and end positions in the protein sequence, modifications applied, the number of missed cleavages and the amino acid sequence of the peptide.

  6. When doing MS experiments there is a possibility that the samples may contain other proteins than the one you are studying, for example, keratin or parts of the enzyme used for digestion. To avoid disturbances due to these non-relevant peptides you can add a filter that will filter out m/z values that may come from such contaminants. To do this: on the "Edit menu", select "Filter(s)". This will open a window where you can select allready created filers or create new ones. Select the filter named "TrypsinNoise" and click on "Update". The selected filter will then be added to the list of theoretical data (for example row 16). Close the theoretical data window (and be sure to select "Yes" when asked about saving). Then click on "Next>".

  7. The third step is the importing of experimental data. Again you have two choices; either import new experimental data files or select one or more from the list of allready available data files. In this tutorial we will start by importing new experimental data. Click on the "Import Experimental Data" button. A new window then presents three choices: Delimited text file, XML file and Cut and Paste. Delimited text file is selected by default. Keep this choice and click on "Next >".

  8. A new window where you can select the file details then appears. Click on the folder icon next to the "File" text field and select the file "bsa-10pmol _0001-Spec.pkt" from the list. Preview of the selected file is then shown, both the raw text file and the imported version. Make sure that the correct column numbers are selected for the mass and the intensity (in this case it should be 2 and 7), and click on "Next >" to continue.

  9. The last import window then appears. Insert the correct protein name (in this case "BSA"), make sure that the correct enzyme is selected (in this case "Trypsin") and insert any comments if wanted. Click on "Edit List" next to the "Considered modifications"-frame. A window with the available modifications appears. Select C-am, gK, oM and pyrE, click "Add >" and "OK". The modifications selected are those expected in the given experiment. This list may vary from experiment to experiment. Finally click on "Import". (For this tutorial the remaining experiments are allready imported, so on the "Do you want to import more data?"-question, click on "No".)

  10. The experimental data is then imported into MassSorter, inserted into the list of available experimental data files and selected. To see the contents of the file, right click on the given row and select "Preview Experimental Data File" from the popup menu.

  11. During the import procedure described in above, it is possible to manually edit the data, e.g., to remove peaks that the user has recognized as noise, or to add a peak that the spectrum analysis program has not recognized. To delete: Mark the row (it becomes blue), go to the "Edit" menu and select "Delete row". Alternatively, it can be deleted by "ctrl+D". To add: Mark the row above where you want to insert a new row, go to the "Edit" menu and select "Insert row after", alternatively use "ctrl+R". An empty row is inserted, and you can put in the relevant m/z value (remember to use "." as decimal point). None of the other cells need to be filled in.

  12. Close the experimental data window to get back to the Project wizard. From the list of available experimental data files, select the three remaining experiments on protein "BSA" and click on "Next >".

  13. The final step has two purposes. Here you get an overview of the data you have selected to be included in the Project and you also have to choose the accuracy to be used for the comparison of theoretical and experimental data. For this experiments select the accuracy type as "ppm" and an accuracy value of 100. Then click "Finish" to complete the creation of the Project.

  14. The main view of a Project is a spread sheet containing all the comparisons of the experimental and theoretical peptides. The logic behind the spread sheet is as follows: Each experimental peptide's m/z value is first compared to the theoretical m/z values. If a match is found within the given accuracy limit, the program checks to see if the given theoretical peptide is modified. If it is, the modification(s) also has to be possible/realistic for the given MS-experiment. If the modification(s) is possible in the given MS-experiment, or the theoretical peptide is not modified, the two peptides are considered "equal" and put on the same line in the table. Row 3 (unmodified) and row 2 (modified) are examples of this. The first 7 columns are data from the theoretical peptide. Then follows three columns per MS experiment. The second column per MS-experiment contains the accuracy between the theoretical and experimental m/z values. As you can see the two mentioned rows are colored green. An experimental m/z value may also match a "filter-mass". These are colored grey, see row 15 for an example.

    If an experimental m/z value does not match any of the theoretical m/z values it is compared to the m/z values from the other MS experiments if any, and placed on the same row if they are within the selected accuracy limit. These are colored yellow, see row 8.

    The spread sheet can also color-code the experimental values according to the detected intensities. Select "Intensity grading" on the "View" menu. The experimental values are then divided into three groups and each group is given a specified color. The default colors are different shadings of green where the most intense has the darkest shading. The colors used can be altered by selecting "Edit color" on the "View" menu. Before continuing, turn the color-coding back to normal by deselecting "Intensity grading" on the "View" menu.

  15. When comparing the m/z values, it is possible to get more than one match (within the accuracy limit) for a given experimental m/z value. The best match (smallest difference) is automatically selected as a "primary match" and the others are labeled "secondary matches". If the match automatically selected as primary is for some reason wrong, you can select one of the others. First make the secondary matches visible by deselecting "Hide secondary matches" from the "View menu". The secondary matches are colored dark green. Choose one of the secondary matches, i.e. one of the dark green cells, and right click on the corresponding third column of the secondary match. A window appears where you can choose the match you want as a primary match or remove the matches all together. NB: Removing all the matches is irreversible!!

  16. Now that you understand the logics of the spread sheet, it is time to start the real analysis. The goal is to minimize the number of yellow cells. This is the same as maximizing the number of m/z-values from the experiments that we can identify with good confidence. First we will look at the DST in a different way. On the Tools menu select "Report". The information included in the spread sheet is compressed into an html-file where (for each experiment) the matches are divided into different categories; matches with unmodified theoretical peptides, matches with modified theoretical peptides, matches with filter(s) and so on. Other statistics are also shown; like % match (of all the m/z-values in the given experiment, how many match with theoretical values within the given accuracy limit) and sequence coverage. The sequence coverage is also shown on a model of the sequence. The red parts are the covered parts. Underscored residues are residues that may be modified. By right clicking on a covered residue information about the peptides containing the selected residue is shown. (Modification details can be accessed in the same way.)

  17. The Report only contains a 2D model of the amino acid sequence of the protein. A 3D model is available by clicking on the "View as 3D model" link. A file chooser appears where you must select a PDB from which the model is created. The structural information from the PDB file is then coupled with the coverage data from the Report and a 3D model is created. The 3D model uses the same color-coding scheme as in the Report, but can also be extended to coloring modifications, residues and amino acids.

  18. There are many ways to increase the number of matched m/z values. One way is to include lots of modifications in the theoretical digestion and make them all possible in all the MS experiments. This will probably make the digestion and comparison significantly slower and also create many incorrect matches, simply by chance, and much work must be done to find the correct ones.

    A better approach is therefore to only include the modifications that are expected and test for others later. MassSorter includes a database called UniMod (www.unimod.org) that contains data on, at the time of writing, 192 different modifications. To search this database for modifications, right click on one of the yellow masses, select "Modification search", and click on "Search". A list of possible modifications that may explain the unmatched m/z value is shown. The list is created as follows: All the theoretical m/z values between "Search mass + lower limit" and "Search mass - upper limit" are compared to the search mass and the difference is calculated. This difference is compared to the list of mass changes from all the modifications in the UniMod database. If the difference between the 'theoretical m/z value' + 'the mass change of a modification' and the experimental m/z value is within the accuracy limit, we have a possible match. If you click on "Insert into DST" on one of the modifications, it is inserted into the DST, and the row is colored blue. A match inserted in this way can be removed by right clicking on the given mass and selecting "Remove match". When you have finished testing this feature, close the search window.

  19. Another way of increasing the number of indentified m/z values, is to check for "non-theoretical" cleavage sites. When MassSorter digests an amino acid sequence it only cleaves at the theoretically correct sites of the enzyme selected. For example, trypsin cleaves after R and K. When digesting in experiments the enzyme sometimes cleaves at other sites as well or a peptide may be sensitive to chemical cleavage. These two cases, combined or alone, may result in peptides that have one or two terminals that don't match any theoretically digested peptides.

    To search for these kinds of peptides: right click on one of the yellow masses and select "Suggest sequence(s)". A window similar to the ProteinDigester appears. Leave the parameters as they are and click on "Suggest sequences". A list of the possible peptides from the given protein sequence, with non-theoretical cleavage sites, appears. If you click on a row in the table, the selected peptide will be marked as blue in the frame in the upper right. The red parts of this sequence are the already covered parts. After selecting a row, the match can be inserted into the DST by selecting "Insert selected mass into DST" from the "File menu". These matches can be removed by right clicking on the given mass and select "Remove match". When you are done testing this feature, close both the SequenceSuggester and the results windows.

  20. If you want to look for modifications in an experiment and that modification was not included in the theoretical digestion or in the list of possible modifications for the experiment, you have to update both the theoretical and experimental data files. The theoretical data file can be changed by right clicking on the header of the column in the spread sheet labeled "Theoretical" and selecting "View Theoretical Data" from the popup menu. If you want to completely change the data, select "Re-digest" from the "Tools" menu. The experimental data can be altered in the same way by clicking on the column in the spread sheet labeled with the experiment name.

  21. Adding or removing experimental data can be done by "Experimental Data" from the "Edit" menu. This window can also be used to change the order of the experimental data files in the spread sheet.

  22. In addition to the tools for finding the origin of the unmatched peptide masses, MassSorter also includes some statistical tools. MassSorter can calculate three types of statistics: peptide statistics and two kinds of accuracy statistics. The peptide statistics are calculated for all the peptides in the DST and are created by selecting "Peptide statistics" from the "Statistics" sub menu on the "Tools menu". It contains statistics for the following peptide properties: hydropathy, peptide length, cleavage site frequencies and amino acid frequencies.

  23. The first kind of accuracy statistics is calculated by selecting "Accuracy statistics" from the "Statistics" sub menu on the "Tools menu". It contains accuracy statistics for all the peptides in the DST. The last kind of statistics available, is individual accuracy statistics for each experiment. These are accessed by right clicking on one of the accuracy values for the wanted experiment and selecting "Accuracy statistics".

    The accuracy statistics can also be visualized as a plot of the m/z values against the accuracy values. This option is available by either right clicking on an accuracy value in the DST table (single experiment plot) or by selecting "Accuracy plot" on the "Tools -> Statistics" menu (multiple experiments in one plot).

  24. That is the end of the tutorial. If you want more help, click on the help icons in the sub windows.

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