TPP Tutorial v2

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Revision as of 19:25, 13 May 2009

1 Quick Start to data analysis using the TPP
2 Other Resources

Quick Start to data analysis using the TPP

1. Download and install the TPP

To install on your Windows system, please follow this link 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.)

Log into Petunia, the TPP GUI

As a way to verify that the installation was successful, 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.

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 include 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. 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.

4. 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.

First, make sure that Tandem is selected as the analysis pipeline.
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.

5. 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.

We also need the mzML data files we converted in step 4. While this can be accomplished within Petunia, it is easier to use the native Windows file copy. Copy the two mzML files located at C:\Inetpub\wwwroot\ISB\data\demo2009\tandem into the directory C:\Inetpub\wwwroot\ISB\data\demo2009\spectrast. Now we can move on to searching these data:

Mouse-over the Analysis Pipeline menu title in Petunia, and then click on the SpectraST Search menu item to access the SpectraST search interface.
In section 1, select the two mzML data files under demo2009\spectrast.
For section 2, select the NIST_yeast_IT_v2.0_2008-07-11.splib.splib spectral library file located under dbase\speclibs. This is the file you downloaded from PeptideAtlas above.
Finally, for section 3, select the yeast_orfs_all_REV.20060126.fix.fasta sequence database, located under dbase.
Leave the rest of the options on the page at their default values, and click on Run SpectraST to initiate the search.

6. Validation of Peptide-Spectrum assignments with PeptideProphet

PeptideProphet provides statistical validation of search engine results by assigning a probability to each peptide-spectrum match.

Click on the Analyze Peptides tab under the Analysis Pipeline section in Petunia to access the xinteract interface.

xinteract is a general utility that is able to launch several components of the TPP, including PeptideProphet.

Select the two OR2008*.pep.xml files in the directory demo2009\tandem. (You can also choose to run this on the SpectraST results if you prefer). Make sure that there are only two files selected for analysis; you can edit the selections using the checkboxes and Remove button on the right-hand side.
Under PeptideProphet Options, find and select the option to Use accurate mass binning since this is a high-accuracy data.

Optional: Run InterProphet

InterProphet (or iProphet) is a tool that provides statistical refinement of PeptidePropet results.

Under InterProphet Options, select to RUN InterProphet.

Optional: Run ASAPRatio

ASAPRatio is a tool for measuring relative expression levels of peptides and proteins from isotopically-labeled samples (e.g. ICAT, SILAC, etc).

Under ASAPRatio Options, select to RUN ASAPRatio, change Labeled Residues to K and R, set m/z range to include in summation of peak to 0.05, set Specified masses to M 147.035, K 136.10916, and R 166.10941.

Leave all other options set to their defaults, and click on Run XInteract at the bottom of the page to run PeptideProphet.
Once the command finishes running, you can click on the view results link that appears in the Command Status box to view and analyze the results.

7. Visualize LC-MS/MS data using Pep3D

Pep3D is a tool for visualizing LC MS data, along with results from PeptideProphet.

Under the Utilities -> Browse Files section in Petunia, navigate to the demo2009\xtandem directory. (nB. you may already be in that directory.)
Open the PeptideProphet results file by clicking on the [ PepXML ] link next to the file named xinteract.pep.xml. This will launch the PepXMLViewer application.
Click on the Other Actions top-level tab, and then on the Generate Pep3D button. A new window will launch the Pep3D viewer.
Leave the default options (or change to taste) and click on the Generate Pep3D Image button.
After a few moments, you should see two images displayed on the page, one per mzML input file.

8. Protein-level validation with ProteinProphet

ProteinProphet is a tool that provides statistical validation of Protein identifications, and is based on PeptideProphet results.

Click on the Analyze Proteins tab under the Analysis Pipeline section in Petunia to access the ProteinProphet interface.
Select the xinteract.pep.xml file in the directory demo2009\tandem. (You can also choose to run this on the SpectraST results if you prefer). Make sure that there is only one file selected for analysis; you can edit the selections using the checkboxes and Remove button on the right-hand side.
Leave all other options set to their defaults, and click on Run ProteinProphet at the bottom of the page to run ProteinProphet.

Other Resources

You can find a longer, more thorough tutorial at the TPP Tutorial page of our Wiki.

Retrieved from "http://tools.proteomecenter.org/wiki/index.php?title=TPP_Tutorial_v2"

 To install on your Windows system, please follow [http://sourceforge.net/project/showfiles.php?group_id=69281&package_id=126912 this link] 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]].)
+=== Log into Petunia, the TPP GUI ===
+As a way to verify that the installation was successful, 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.
 == 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.
+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 include a search parameters file.
 * Please download the demo data as a zip file from this link (NEED LINK).
-== 3. Log into Petunia, the TPP GUI ==
+== 3. Convert raw data to the mzML format ==
-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'''.
-== 5. Search data with X!Tandem ==
+== 4. 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.
+* First, make sure that ''Tandem'' is selected as the analysis pipeline.
 * 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.
-== 6. Search data with SpectraST ==
+== 5. 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.
+''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'''.
-== 7. Validation of Peptide-Spectrum assignments with PeptideProphet ==
+== 6. Validation of Peptide-Spectrum assignments with PeptideProphet ==
 ''PeptideProphet'' provides statistical validation of search engine results by assigning a probability to each peptide-spectrum match.
 * Select the two '''OR2008*.pep.xml''' files in the directory '''demo2009\tandem'''.  (You can also choose to run this on the ''SpectraST'' results if you prefer).  Make sure that there are only two files selected for analysis; you can edit the selections using the checkboxes and ''Remove'' button on the right-hand side.
 * Under ''PeptideProphet Options'', find and select the option to '''Use accurate mass binning''' since this is a high-accuracy data.
+=====Optional: Run InterProphet=====
+:''InterProphet'' (or ''iProphet'') is a tool that provides statistical refinement of ''PeptidePropet'' results.
+:* Under ''InterProphet Options'', select to '''RUN InterProphet'''.
+=====Optional: Run ASAPRatio=====
+:''ASAPRatio'' is a tool for measuring relative expression levels of peptides and proteins from isotopically-labeled samples (e.g. ICAT, SILAC, etc).
+:* Under ''ASAPRatio Options'', select to '''RUN ASAPRatio''', change ''Labeled Residues'' to '''K''' and '''R''', set ''m/z range to include in summation of peak'' to '''0.05''', set ''Specified masses'' to '''M 147.035, K 136.10916,''' and '''R 166.10941'''.
 * Leave all other options set to their defaults, and click on '''Run XInteract''' at the bottom of the page to run ''PeptideProphet''.
 * Once the command finishes running, you can click on the '''view results''' link that appears in the ''Command Status'' box to view and analyze the results.
-== 8. Visualize LC-MS/MS data using Pep3D ==
+== 7. Visualize LC-MS/MS data using Pep3D ==
 ''Pep3D'' is a tool for visualizing LC MS data, along with results from ''PeptideProphet''.
-== 9. Extra statistical analysis with iProphet and Quantitation with ASAPRatio ==
+== 8. Protein-level validation with ProteinProphet ==
-''InterProphet'' (or ''iProphet'') is a tool that provides statistical refinement of ''PeptidePropet'' results.  ''ASAPRatio'' is a tool for measuring relative expression levels of peptides and proteins from isotopically-labeled samples (e.g. ICAT, SILAC, etc).  Both of these tools can be run from the ''XInteract'' page in ''Petunia''.
-* Click on the '''Analyze Peptides''' tab under the ''Analysis Pipeline'' section in ''Petunia'' to access the ''xinteract'' interface.
-* Select the two '''OR2008*.pep.xml''' files in the directory '''demo2009\tandem'''.  (You can also choose to run this on the ''SpectraST'' results if you prefer).  Make sure that there are only two files selected for analysis; you can edit the selections using the checkboxes and ''Remove'' button on the right-hand side.
-* Under ''PeptideProphet Options'', find and select the option to '''Use accurate mass binning''' since this is a high-accuracy data.
-* Under ''InterProphet Options'', select to '''RUN InterProphet'''.
-* Under ''ASAPRatio Options'', select to '''RUN ASAPRatio''', change ''Labeled Residues'' to '''K''' and '''R''', set ''m/z range to include in summation of peak'' to '''0.05''', set ''Specified masses'' to '''M 147.035, K 136.10916,''' and '''R 166.10941'''.
-* Leave all other options set to their defaults, and click on '''Run XInteract''' at the bottom of the page to run ''PeptideProphet'', ''interProphet'', and ''ASAPRatio''.
-* Once the command finishes running, you can click on the '''view results''' link that appears in the ''Command Status'' box to view and analyze the results.
-== 10. Protein-level validation with ProteinProphet ==
 ''ProteinProphet'' is a tool that provides statistical validation of Protein identifications, and is based on ''PeptideProphet'' results.