TPP Tutorial v2
From SPCTools
Contents |
Quick Start to data analysis using the TPP
1. Download and install the TPP
To install on your Windows system, please follow this link [1] and click on "TPP_Setup_v4_2_JETSTREAM_rev_1.exe". Select "Run" if prompted, and follow the instructions on the wizard. (For a detailed installation guide, please read our Windows Installation Guide.)
2. Download and install the test data and database
For this demo, we will be using a SILAC-labeled Yeast dataset, comprised of 2 runs on a high mass-accuracy Orbitrap instrument, along with a Yeast database appended with decoys. We also a search parameters file.
- Please download the demo data as a zip file from this link (NEED LINK).
- Unzip (unpack) this file; you should find 3 files.
- Copy or move the yeast_orfs_all_REV.20060126.fix.fasta file into the folder C:\Inetpub\wwwroot\ISB\data\dbase
- Copy or move the two data files (OR20080317_S_SILAC-LH_1-1_01.raw and OR20080317_S_SILAC-LH_1-1_11.raw) as well as the tandem parameters file into the folder C:\Inetpub\wwwroot\ISB\data\demo2009\tandem . Create this last folder if necessary.
3. Log into Petunia, the TPP GUI
Log into Petunia by double-clicking on the Trans-Proteomic Pipeline flower icon on your Desktop or through the Start menu. Alternatively, you can open a browser window into the following URL: http://localhost/tpp-bin/tpp_gui.pl . You can use the credentials guest and guest as user name and password to log in.
Once you are in the Home page, please select Tandem as the analysis pipeline, just below the Welcome message.
4. Convert raw data to the mzML format
We have developed the TPP (and dozens or related tools) to read mass-spec data from a common, open data format. We must therefore first convert the proprietary raw data to this format, called mzML.
You can only do this data conversion step if the TPP is installed on a machine that also contains an installation of Bioworks or Xcalibur, as the converter needs to use vendor software libraries to read the raw data. If your machine does not have either of these, you can download the mzML files from this link (NEED LINK) and skip to the next step.
- Mouse-over the Analysis Pipeline (Tandem) portion of the navigation links near the top of the page; a pop-up menu should appear. Select the mzML item in this menu.
- Click on the Add Files button in the first section; the File Chooser window will open.
- Click on the demo2009 directory link on the right portion of the page. Then select tandem.
- Select both raw data files by clicking on the checkbox next to each, then on the Select button at the bottom. This should return you to the mzML page along with a confirmation of the files that you just selected.
- Leave the Conversion Options unchecked.
- Click on Convert to mzML; a wait page should appear.
- The Command Status box should automatically change color to orange when the conversions are done.
5. Search data with X!Tandem
A custom version of the popular open-source search engine X!Tandem is bundled and installed with the TPP. It has been modified from the original distribution by adding the K-Score scoring function, developed by a team at the Fred Hutchinson Cancer Research Center.
- Click on the Database Search tab under Analysis Pipeline to access the X!Tandem search interface.
- Click on Add Files and select the two mzML files present in the demo2009\tandem directory as input files for database searching.
- Similarly, choose the Tandem parameters file called tandem-params.xml located in the same directory.
- Lastly, select a sequence database to search against. Navigate up to the dbase directory in the File Chooser, and select the database file yeast_orfs_all_REV.20060126.fix.fasta.
- Start the search by clicking on Run Tandem Search.
Convert results to PepXML
Since each search engine provides results in different ways, the TPP requires that they be converted to a common format for downstream processing. This is the PepXML format, and can the conversion can be effected via the pepXML tab of the Analysis Pipeline.
- Choose the two OR2008*.tandem files in the demo2009\tandem directory; these are the X!Tandem search results.
- Click on Convert to PepXML.
6. Search data with SpectraST
SpectraST is a search engine that compares acquired spectra against a library of pre-identified spectra to which peptide sequences have been assigned. In order to conduct the search, we must first download the appropriate spectral library.
- Go to the Home page, and switch the pipeline type to SpectraST.
- Under the SpectraST Tools section of the navigation menu, select the Download Spectral Libraries menu item.
- You are now at at page that shows a list of spectral libraries available at PeptideAtlas, along with locally-installed/downloaded ones. Select the NIST_yeast_IT_v2.0_2008-07-11.splib.zip (yeast ion trap) library on the right pane, and click on Download Selected Libraries.
tbc...
