Tutorials

ProteoSuite is still under development, however, we have produced a quick tutorial to try out our beta version. This tutorial describes the different sections of the software and presents an example for running iTRAQ on ProteoSuite.

 

1. Installation

2. Getting started

3. ProteoSuite GUI Tools - An overview

3.1. Visualization of MS spectra

3.2. Visualization of Chromatograms

3.3. Visualization of raw data as 2D plots

3.4. Visualization of PSI-Standard formats

4. Running a Pipeline

4.1. Running an iTRAQ experiment

5. Other tools and utilities

5.1. Convert mzML to MGF

 

 

 

1. Installation

The following instructions apply for ProteoSuite 0.2.2 and above.  Use one of the following categories depending on your operating system. Before installing ProteoSuite make sure you have read the software and hardware requirements section.

 

Windows

  • Save the ProteoSuite-X.X.X.zip file into one of your local drives.
  • Unzip the program in any of your local drives. E.g. C:\ProteoSuite-X.X.X
  • Locate and execute (double click) the jar file (proteosuite-X.X.X.jar) to start ProteoSuite. You can also use the startup.bat file which allows to customise the RAM memory used in ProteoSuite [i.e. 1024MB (1GB) RAM is set up as default].

         Tip: You can create a shortcut to this file and place it into your desktop.

 

Linux

  • Save the ProteoSuite-X.X.X.zip file into one of your local drives. E.g. /home/useraccount/Downloads
  • Unzip the program in any of your local drives. E.g. /home/useraccount/ProteoSuite-X.X.X/
  • Create a shortcut (launcher) to ProteoSuite and place it into your desktop.

i.e. Right click on your desktop. Then Create Launcher. Enter the following information for the launcher: Type: Application Name: ProteoSuite Command: java -jar /home/useraccount/ProteoSuite-X.X.X/proteosuite-X.X.X.jar Comment: ProteoSuite

  • Double click on the launcher.

 

OSX

  • Save the ProteoSuite-X.X.X.zip file into one of your local drives.
  • Unzip the program in any of your local drives. E.g. /Documents/ProteoSuite-X.X.X
  • Locate and execute (double click) the jar file (proteosuite-X.X.X.jar) to start ProteoSuite.

 

Tip: You can create a shortcut to this file and place it into your desktop.

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2. Getting started

To run ProteoSuite simply double click on the proteosuite-X.X.X.jar file or on your desktop icon. All files needed to run ProteoSuite have been included in the zip file. A default configuration file (config.xml) has been also included (Note: You can also customise all these paramaters within ProteoSuite).

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3. ProteoSuite GUI Tools - An overview

ProteoSuite has been designed in an intuitive Graphical Unit Interface (GUI) mode.  We have developed the software in a user-friendly format in order that bench scientist can easily analysed their data. 

 

The framework

 

ProteoSuite consists of 5 main sections as shown in Figure 1:

1) The Project Pipeline that will be run (e.g. SILAC, iTRAQ, Label free, etc.),

2) a file container for the raw (.mzML, .MGF), identification (.mzid, .xml) and the quantitation (.mzq) files used in the pipeline,

3) the visualizer which includes (i) a MS spectra viewer, (ii) a Chromatogram Viewer and (iii) a 2D plot viewer,

4) a window to visualize metadata associated to the raw|identification|quantitation files,

5) and a log window for commands performed in ProteoSuite and a window for the examination of raw data.

 

Figure 1. Screenshot of the ProteoSuite GUI.

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3.1. Visualization of MS spectra

To visualise MS spectra data do the following:

  • Click File -> Import File.
  • Locate the file using the File Chooser. Make sure that you have selected Files of type: Raw Files (*.mzML, *.mzML.gz, *.mgf).
  • The loading process will take some time depending on the size of your file (~3-4 minutes for a 2.5GB mzML in a "normal" PC).
  • Once you have loaded your file, the access to each individual spectrum will be very fast. Click on the "Raw Files" tabsheet (left side).
  • The different scans will be displayed in the right side on the "mzMLView" tabsheet (right side).
  • Click on a particular scan to see the Spectrum View (Fig. 2) and Raw Data.

Figure 2. Example of MS spectrum view.

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3.2. Visualization of Chromatograms

To visualise the Chromatogram do the following:

  • Once you have loaded your file (See first three steps in 3.1 Visualization of MS spectra), click on the "Show Chromatogram"  button in the "mzML View" tabsheet (Fig. 3).

 

Figure 3. Example of chromatogram view.

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3.3. Visualization of raw data as 2D plots

To visualise the raw data as 2D plots  do the following:

  • Once you have loaded your file (See first three steps in 3.1 Visualization of MS spectra), click on the "Show 2D Plot" button in the "mzML View" tabsheet (Fig. 4).

Figure 4. Example of 2D View.

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3.4. Visualization of Mascot XML Files

ProteoSuite also includes a visualizer for Mascot XML identification files. To visualise identifcation data do the following:

  • Click File -> Import File.
  • Locate the file using the File Chooser. Make sure that you have selected Files of type: Identification Files (*.mzid, *.xml).
  • The different identifications will be shown in the "Mascot XML View" tabsheet in the right side.

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4. Running a Pipeline

ProteoSuite has been designed to simplify user interaction. To run a particular pipeline, please use the following workflow as shown in Figure 5:

Figure 5. Typical workflow for a quantitative analysis pipeline in ProteoSuite.

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1. Load your raw file(s) (E.g. .mzML, .mgf). This will take sometime while the program constructs an index to access each element.

2. Load your identification (.mzid, .xml from mascot) file(s).

3. Select the technique used in your experiment (e.g. iTRAQ, SILAC, Label Free, etc.). In the latest release (0.2.3) we are only able to support iTRAQ but other techniques will be released soon.

4. Output results are available as mzQuantML (.mzq) and/or comma-separated value (.csv) file format. 

 

 

4.1. Running an iTRAQ experiment

 

1. Add your raw files (e.g. Import File -> ksl_1_10.mzML, available at the Datasets for test section)

2. Add your identificaton files (e.g. Import File -> Mascot_ksl_1_10.mzid, available at the Datasets for test section)

3. Select iTRAQ as the technique that will be used.

4. Select the ouput format (e.g. mzQuantML (mqz) or CSV file).

 

Advanced Settings

You can modify the parameters that have been set up by default for iTRAQ.

  • To do this go to: Project->Set Quantitation Parameters->iTRAQ/TMT. (You can also restore the values to default settings).

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5. Other tools and utilities

ProteoSuite also incorporates other tools that may be useful for the Proteomics community, for example, we have developed some utilities that can convert mzML files into Mascot Generic Format (MGF) files, etc.

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5.1. Convert mzML to MGF

When writing quantitation software we often require linking back the raw data for peptide identifications. Therefore, we need to send some parameters such as scan number and/or retention time in order that the search engine can keep the value. To do this we have produced a java plugin which converts mzML into the Mascot Generic Format (MGF) file and set the parameters in the scan_title tag.

To convert mzML raw files into MGF files do the following:

  • Click Tools -> Converters -> mzML to MGF.
  • Select the ouput folder, this can be any local directory e.g. D:\Data (Windows), /Documents/Data (OS X), etc..
  • Add the mzML files to convert.
  • Click Convert to start the process.

 

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Copyright © ProteoSuite 2011-2013   Project Funded by the British Biotechnology and Biological Sciences Research Council, under the Bioinformatics and Biological Resources Scheme:
http://www.bbsrc.ac.uk/funding/opportunities/2009/bioinformatics-biological-resources-fund.aspx