# The main program window

graphical user interface GUI

Proteomics data explorations, with &i2mcq;, entail, for a large part, the following steps:

- Configuration of the &xtandem; external software that runs the database searches (producing peptide vs mass spectrum matches&emdash;PSMs&emdash;, leading to the peptide identifications and ultimately to protein identifications);

- Configuration of the protein database files (both the organism-specific protein databases and optional contaminant-containing databases);

- Loading of the mass spectrometry data acquisition files (the mzML format is recommended);

- Running &xtandem; from inside of &i2mcq;;

- Loading of the identification results produced during the previous step;

  &i2mcq; can also handle peptide vs spectrum matches data (peptide identification data) from other software with the following formats:

  - mzIdentML;

  - pepXML;

  - Mascot DAT files

- Relentless scrutiny of the peptide identification results. Optional modification of the results;

- Protein inference, that is, protein identification on the basis of the peptide identifications. &i2mcq; implements a protein grouping algorithm, as described in , that leads to consolidated protein identifications. The program has an interface geared towards the tweaking of the protein grouping process so as to let the user in full control of the stringency with which the protein identifications list is ultimately generated.

In this chapter, &i2mcq;'s main window's user interface is described in detail, in particular in the way it is a starting point for the main tasks briefly mentioned above.

## Starting a new &i2mcq; working session

To start a session, run &i2mcq; and the main program windows shows up as described in .

Main program window

The main program window contains three buttons described in detail in the text.

The main program window contains three buttons that start the following main tasks:

- Run X!Tandem identifications. See .

- Load identification results (mzIdentML, pepXML, Mascot, X!Tandem). See .

- Load an i2MassChroQ project. See .

## Running &xtandem; identifications

To run &xtandem;-based identifications, click onto the Run X!Tandem identifications button. This triggers the opening of the window pictured in .

&xtandem;-based identification configuration

The configuration of a &xtandem; run is performed in this configuration window (see text for details).

The configuration of an &xtandem; run entails defining the following:

- Configure the X!Tandem execution: This setting allows one to specify the path to the &xtandem; software program. The version of the program, if found, is displayed below (in this case, Alanine 2017.2.1.4). This feature is useful when the user wants to test multiple versions of the &xtandem; software.

- Run X!Tandem through HTCondor: Only check the box if running &xtandem; over the network on a server supporting HTCondor

  See .

  .

- Choose presets: This setting defines the parameters that &xtandem; must use. Either load already known presets from the drop-down list widget or edit them (or create a new set) by clicking onto the Edit button. Note that to load an existing presets file, it might be necessary to point &i2mcq; to the directory that contains the presets file. Use the folder icon for this, as visible in .

- Choose database files: Add protein database files in the FASTA format. There must be at least one protein database that contains all the known proteins for the organism of interest (there might be as many such database files as necessary) and optionally protein databases containing known contaminant proteins (there might be as many such database files as necessary). Click onto the Clear list button to clear the database files list and start anew if an error occurred (it is not possible to remove files one at a time).

- Choose MS data files to process: Add the mass spectrometry data files (mzML or mzXML format) to be processed by the &xtandem; software. As many files as necessary might be added in the list.

  When using Bruker timsTOF data, click onto the Add Bruker timsTOF folders button to select folders containing this kind of data. Bruker timsTOF data come as two files that must sit in the same directory.

- Output directory: This setting specifies the directory into which new files output by the &xtandem; process need to be created. &xtandem; produces identification results in files in an XML format that &i2mcq; reads during a later step.

- Number of threads: This setting defines the maximum number of execution threads that &xtandem; might be using during its run.

  Although &i2mcq; sets that number of execution threads to 1, it is beneficial to set that number to the highest value possible.

## Setting the &xtandem; Run Presets

The Edit button of the Choose presets group box described above triggers the opening of a dialog window where the user might configure in the most detailed way the &xtandem; parameters. That dialog window is pictured in . Only the Spectrum tab is shown, but the interface is similar for all the other ones.

&xtandem; presets configuration window (Spectrum tab)

The configuration of the &xtandem; presets is performed in this configuration window. This window has its Spectrum tab selected. Each parameter is associated to a manual page

The help texts are extracted automatically from the &xtandem; software documentation.

that can be displayed by clicking on the interrogation mark button next to it. It is possible to load existing presets from file or to create brand new ones.

### Loading existing presets configurations from file

It is possible to load existing &xtandem; presets (which is useful in particular if the samples most often come from the same instrument using the same configuration). To this end, first point &i2mcq; to the right directory that contains the presets file of interest (click onto the folder icon at the top right corner of the window shown in ). The presets files in the chosen directory are automatically detected and listed in the drop-down list widget. At this point, select from that list the file of interest and click onto the Load button.

It is compulsory to click onto the Load button to confirm loading of the presets file contents, because these are not updated upon choosing the file name from the drop-down list only.

### Creating new presets configurations

It is possible to create a new presets file by clicking onto the New button. This opens an input dialog window for the user to provide a new file name (the edit widget is preset with the currently loaded file's name suffixed with _copy).

One interesting feature of the new presets file creation process is that, if presets are already loaded, &i2mcq; copies the currently displayed settings to the new file. From there, it is possible to create a variant &xtandem; presets file, which eases the exploration of the right &xtandem; parameters for a given sample data set.

### Actual &xtandem; presets configuration

The dialog window pictured in contains a number of tabs where various aspects of the &xtandem; run settings are handled. Each parameter's documentation can be seen on the pane on the right hand side of the window by clicking onto the question mark button next to it. These manual pages are authoritative because they are taken from the &xtandem; software package with no transformation whatsoever.

Once the configuration has been performed, click onto the OK button. If the parameters were modified, &i2mcq; asks if they should be stored in the file.

### Running a properly configured &xtandem; process

Once the &xtandem; settings configuration dialog window has been closed, it is possible to run &xtandem; from inside &i2mcq; by clicking onto the Run button at the bottom of the window pictured in .

While the computation is carried over, the program shows the feedback dialog window pictured in .

&xtandem; run feedback to the user

The text in this feedback dialog window is getting incrementally printed all along the computation.

Once the computation is finished, the feedback dialog window closes and the user is returned to the main program window () albeit with a message shown in .

&xtandem; run finished message to the user

The &xtandem; run is now finished. Click the OK button to access the main program window.

From the main program window, it is possible to open the &xtandem; results file(s) located in the output directory configured above. There are as many output files (XML-based format, and xml extension) as there were mass spectrometry data files to process. The loading of the results files is carried over by first clicking the button labelled Load identification results (mzIdentML, pepXML, X!Tandem). The process is described in .

## Loading the Protein Identification Results

The loading of identification results comes with a minimal set of configuration required to instruct &i2mcq; on the way to handle contaminant proteins, for example. This process is pictured in and is described in the following section.

Configuration of the loading of the identification results

Loading identification results comes with some configuration that is described in the text.

### Identification Data Loading Configuration

- Results handling mode: there are two possibilities:

  - Combine: in this mode, all the identification results coming from different identification results files are merged into a single set. That single set is the basis for the protein inference step and the identified proteins are listed into a single protein list window.

    Selecting a particular identification results file's data set

    When loading multiple identification results files in Individual mode, the selection of any given identification results file is performed by selecting its name from the drop-down list widget and by clicking onto the View protein list button. Note that some metadata about the identifications are updated beneath the drop-down list widget.

  - Individual: in this mode, the identification results coming for various files are kept separated. Thus, the identification results coming from each file are used for a separate protein inference step. The identified proteins list is thus displayed for each single file in turn. The selection of the file for which the protein list needs to be displayed is done via the main program window that changes its appearance:

    Selecting a particular identification results file's data set

    When loading multiple identification results files in Individual mode, the selection of any given identification results file is performed by selecting its name from the drop-down list widget and by clicking onto the View protein list button. Note that some metadata about the identifications are updated beneath the drop-down list widget.

    Right after having selected an identification results file, click onto the View protein list to display the protein identifications list. That list has been obtained by performing the protein inference on the file's protein identification results (see ). The window that opens up will be described later (see ).

    It is possible to open multiple protein list windows, each showing the identifications from a different file: maintain the Ctrl keyboard key pressed while clicking onto the View protein list button.

- Choose results files: by clicking onto the Add files button, the user is provided a file selection dialog window from which any number of protein identification results files might be selected for loading.

  Note that it is possible to list all the opened protein identification results files by clicking onto the View MS identification list button. The window that opens up will be described later (see ).

- Contaminants: there are two possibilities here.

  - Contaminants files: when this radio button widget is selected, the list of contaminant proteins will be loaded from the files selected by clicking onto the Add files button.

  - Contaminant regular expression: when this radio button widget is selected, a text edit widget is shown, replacing the widget listing the contaminants database files. In this text edit widget, the user may enter a regular expression to match the accession number field of the protein databases that were used for the protein identification step. In this situation, the user must use specially crafted protein databases in which the contaminant proteins were tagged on the accession number using a particular text pattern. That particular text pattern is then matched against the Contaminant regular expression that the user enters in the text edit widget.

- Contaminant removal mode: there are two possibilities. The contaminant removal is the process by which, when identified proteins match proteins in the contaminants realm (either from the contaminants dabase files or as determined using the regular expression), they are disregarded for the later protein visualization steps.

  - Protein list: in this mode, as soon as a protein identification loaded from a protein identification results file matches a contaminant protein, it is disregarded.

  - Groups: in this mode, the protein inference process goes all the way through to the determination of the protein groups (see ). When protein groups have sub-groups that contain a contamimant protein, then the whole group is disregarded. This might appear drastic, but our experience is that most often, the sub-groups in a group do identify proteins belonging to the same family. Therefore, if one protein is contaminant, all the other proteins in the group are supposed to be such also.

- Peptide and protein filters: this group box widget holds some parameters that configure the way protein inference is to be performed.

  - Peptide threshold on: there are two possibilities:

    - E-value: all the PSMs having an expectation value higher than that value are disregarded. Enter the value in the spin box widget labelled Peptide E-value. A typical value for the &xtandem; engine is 0.05. When more stringent results are desirable, setting 0.02 should yield satisfactory results. See of a detailed explanation of the E-value computation.

    - FDR (false rate discovery): the PSMs are disregarded if their FDR value does not match this parameter. Enter the value in the spin box widget labelled Peptide FDR. A typical setting is 1%.

      Using FDR is most useful when the identification results come from a database searching engine that does not compute an E-value. However, it does only work if the searching step was performed also on a decoy database. In &xtandem; the decoy database is crafted by reversing the peptide sequences. In this case, when proteins are identified on the basis of the reversed peptide PSM, then the protein identity is tagged with the reversed string, which might be used with the Contaminant regular expression setting defined earlier.

  - Number of peptides per protein: this is the minimal required number of peptides that must be identified as belonging to a given protein in order to consider that protein identity as a valid one. These peptides have to be from non-contaminant proteins, of course.

  - Overall samples: when checked and if multiple identification results files are to be loaded, then the Number of peptides per protein requirement might be fulfilled by looking for peptides in all the loaded files. For example, if one results file provides one peptide for a protein identification and another file provide another peptide (different from the first one) to identify the same protein, and if the Number of peptides per protein is 2, then the protein is considered as a valid protein. If not checked, that number of peptides requirement must be fulfilled by looking into each results file separately. This last setting is more stringent. A typical value for this setting is 2.

    This setting needs to be checked in at least one case: when a complex peptidic mixture is separated by ion chromatography (typically on an SCX&emdash;strong cation exchange&emdash;resin) and the different fractions are analyzed by bottom-up proteomics. The peptides coming from a given protein might be located in different fractions, and thus in different protein identification results files!

  - Protein Evalue: threshold above which a protein identification is disregarded (see ).

  - Protein Evalue (log10): convenience spin box widget for the user to easily set the protein E-value.

  - Pep repro: if set to 1, a peptide, to be accounted for, needs to be found in one protein identification results file. If set to a greater number, then that peptide needs to be found in that number of results files. This setting sets more stringent protein identification conditions each time it is incremented.

### Displaying the MS Identifications List

Displaying the MS identifications list (first columns)

This window displays a list of all the files that were involved in the &xtandem; run (first columns).

Displaying the MS identifications list (last columns)

This window displays a list of all the files that were involved in the &xtandem; run (last columns).

### Saving &i2mcq; projects

Once exploration and optional modication of the identification data have been performed, the user can save the resulting data set into a &i2mcq; project by selecting the Save project menu item of the File menu in the main program window (the extension of the file name typically should be `xpip`). See for loading such a project.

## Loading &i2mcq; projects

Loading of &i2mcq; project files (file of `xpip`) extension) is only possible if the user has previously

- Loaded identification results;

- Saved the data to an &i2mcq; project file using the Save project menu item of the File menu in the main program window.