8 Specific procedures for the timsTOF line of instruments

It is possible to load Bruker timsTOF data right in the i2MassChroQ program's graphical user interface, as shown in the window pictured in Figure 3.2, “X!Tandem-based identification configuration”. The very first specific step to take in this case is to select the data files by clicking the Add Bruker timsTOF folders.

The Add Bruker timsTOF folders button lets the user choose the Bruker data directory (.d extension) and then asks if the data to be loaded are in the TDF or MGF format (see Figure 8.2, “File format selection dialog for Bruker timsTOF data”).

Indeed, the MGF file generated by the Bruker software is automatically installed in the .d extension data directory.

When handling Bruker timsTOF data, two file formats are available: MGF and TDF. See text for details.

Figure 8.2: File format selection dialog for Bruker timsTOF data #

The DataAnalysis software from Bruker allows one to export proteomics MS/MS data into MGF format files (Mascot generic format). Their native data format, though, is the TDF format. It is important to keep in mind that the MGF format only stores MS/MS spectral data, no MS data. By using this format, i2MassChroQ and MassChroQ won't be able to access MS data, which are required in a number of situations, in particular when extracting ion currents for given m/z ratios, for example, or for area-under-the-curve quantitative proteomics. It is thus always recommended to use the native TDF format whenever available.

When loading Bruker timsTOF data right into i2MassChroQ as described above, the software performs some under-the-hood operations that the user might want to be aware of. The hidden operations are unveiled in the following sections, as they involve command line programs shipped along with i2MassChroQ that might be of interest to the user.

X!Tandem needs the mass spectrometric data that it uses for the database searches to be in the mzXML format. For this very reason, i2MassChroQ cannot work by harnessing the capabilities of X!Tandem starting from Bruker timsTOF data. These data need to be converted to an mzXML file before they can then be fed to X!Tandem.

In order to be able to store a mzXML file on disk, the user may convert timsTOF data files (TDF or MGF) to mzXML using the mzxmlconverter program that is shipped along with i2MassChroQ. This program is a command line program that takes a data file in input and that writes a mzXML file in output. The command line syntax is easy, as picture in Figure 8.3, “Converting mass data files to mzXML data files”.

To obtain help about the program, run the following:

$ ^[16] <path_to>/mzxmlconverter --help RETURN

Files of any format handled by ProteoWizard or files from the Bruker's timsTOF line of instruments can be converted to mzXML using mzxmlconverter. Conversion from timsTOF format to mzXML is performed entirely by our own software.

Figure 8.3: Converting mass data files to mzXML data files #

Warning: The mzXML file format does not contain mobility data from the timsTOF data files

It is important to grasp that the mzXML file that is generated by mzxmlconverter does not contain all the mass data that are contained in the Bruker's timsTOF data files. When loading the produced mzXML format files in i2MassChroQ, the X!Tandem program will be able to perform peptide and protein identifications. Later, however, when the user will try to activate features in i2MassChroQ that require the original data, the software will not be able to provide the expected results, like XIC reports or ion mobility values, because it won't have access to the original data.

The mzxmlconverter program is practical because it allows storing the mzXML file on disk and loading it in i2MassChroQ for X!Tandem to consume it for the identifications. However, as stated in the warning above, that mzXML file has not a full copy of the data in the original mass spectrometry data file (be that a mzML, or MGF, or TDF file). i2MassChroQ has a solution for this problem: using an integrated workflow to convert the original data file to mzXML, make X!Tandem use it, write out the X!Tandem results file and finally rewrite that file into a new version by adding a connection between the X!Tandem run results and the original mass data file. In this way, when the user will activate features in i2MassChroQ that need accessing the original mass data file, the expected results will be effectively displayed. This process is described in detail in the next section.

In order to seamlessly use Bruker timsTOF data in the context of performing X!Tandem identifications and later MassChroQ-based quantifications, the tandemwrapper program is made available to users who want to perform database searches using the command line interface. The tandemwrapper program performs an under-the-hood file format conversion as described in the previous section before automatically feeding the generated mzXML file to X!Tandem. After X!Tandem has produced its results file, that file is rewritten by tandemwrapper in such a manner that the connection with the original mass spectrometry data files is reinstated for further use in the i2MassChroQ graphical interface.

There is, however, a way to convert Bruker timsTOF data files to mzXML using a standalone program, called tandemwrapper, that is shipped along with i2MassChroQ. The tandemwrapper program is a command line program that takes as input a XML configuration file. The XML file is most similar to the configuration file that X!Tandem uses.

To obtain help about the program, run the following:

$ <path_to>/tandemwrapper --help RETURN

A typical tandemwrapper input configuration file is shown below:

  <?xml version="1.0" encoding="UTF-8"?>
  <bioml label="example-tandemwrapper-mass-data-file.mzxml">
    <note type="heading">Paths</note>
    <note type="input" label="list path, default parameters">/full_path_to/xtandem-presets-file.xml</note>
    <note type="input" label="list path, taxonomy information">/full_path_to/database.xml</note>
    <note type="input" label="spectrum, path">full_path_to/mass-data-file.mzxml</note>
    <note type="heading">Protein general</note>
    <note type="input" label="protein, taxon">usedefined</note>
    <note type="heading">Output</note>
    <note type="input" label="output, path">/full_path_to/tandemwrapper-output.xml</note>
  </bioml>

The XML configuration file that is provided to tandemwrapper on the command line is a replicate of the file that X!Tandem itself expects. That file is shown in Figure 8.4, “Configuration file for tandemwrapper”.

The tandemwrapper program takes as input a configuration file that is most similar to the configuration file that is fed to X!Tandem.

Figure 8.4: Configuration file for tandemwrapper #

The following elements need an explanation:

The configuration file that indicates where the FASTA protein database files are located, that is referenced in the tandemwrapper input configuration file is shown in Figure 8.5, “Configuration file pointing at the FASTA protein databases”.

This file tells tandemwrapper the location of the FASTA protein databases required when X!Tandem will actually perform the searches.

Figure 8.5: Configuration file pointing at the FASTA protein databases #

The tandwrapper program performs the following tasks in sequence: