GProX

TUTORIAL

Revision: May 19th 2011, KR

Contents

Preface ..................................................................................................................................................................................................................... 2

1 Data ............................................................................................................................................................................................................... 3 2 Installation ..................................................................................................................................................................................................... 3

2.1 Install required components ............................................................................................................................................................ 3 2.1.1 Install R ......................................................................................................................................................................................... 3

3 Data Import ................................................................................................................................................................................................... 3 3.1.1 Step 1 ............................................................................................................................................................................................ 3 3.1.2 Step 2 ............................................................................................................................................................................................ 4 3.1.3 Step 3 ............................................................................................................................................................................................ 4 3.1.4 Step 4 ............................................................................................................................................................................................ 4

4 output ............................................................................................................................................................................................................. 5 4.1 Select Graphical formats .................................................................................................................................................................. 5 4.2 Locate output ..................................................................................................................................................................................... 5

5 Data Browsing .............................................................................................................................................................................................. 5 5.1 Columns ............................................................................................................................................................................................. 5

5.1.1 Rename ........................................................................................................................................................................................ 5 5.1.2 Move .............................................................................................................................................................................................. 5 5.1.3 Delete ............................................................................................................................................................................................ 6 5.1.4 Insert ............................................................................................................................................................................................. 6

5.2 Rows ................................................................................................................................................................................................... 6 5.2.1 Find ................................................................................................................................................................................................ 6 5.2.2 Delete ............................................................................................................................................................................................ 6 5.2.3 Move .............................................................................................................................................................................................. 7 5.2.4 Sort ................................................................................................................................................................................................ 7

5.3 Data table sheets .............................................................................................................................................................................. 7 5.3.1 rename .......................................................................................................................................................................................... 7 5.3.2 Close ............................................................................................................................................................................................. 7 5.3.3 Open .............................................................................................................................................................................................. 8 5.3.4 delete ............................................................................................................................................................................................. 8

5.4 Browse Functions ............................................................................................................................................................................. 8 5.4.1 Plot proteins ................................................................................................................................................................................. 8 5.4.2 Retrieve database info ................................................................................................................................................................ 8

6 Navigating Graphics .................................................................................................................................................................................... 9 6.1.1 select object to display ................................................................................................................................................................ 9 6.1.2 Delete ............................................................................................................................................................................................ 9 6.1.3 Rename ........................................................................................................................................................................................ 9 6.1.4 New folder..................................................................................................................................................................................... 9 6.1.5 Move .............................................................................................................................................................................................. 9

7 tools ............................................................................................................................................................................................................. 10 7.1 Transformation ................................................................................................................................................................................ 10

7.1.1 Log2 transformation .................................................................................................................................................................. 10 8 Plot data distributions ................................................................................................................................................................................ 10

8.1 Box plot ............................................................................................................................................................................................ 10 8.2 Scatter Plot ...................................................................................................................................................................................... 10 8.3 Histogram ......................................................................................................................................................................................... 10 8.4 Heatmap ........................................................................................................................................................................................... 11

9 Clustering .................................................................................................................................................................................................... 11 10 Retrieve database information ................................................................................................................................................................. 11

10.1.1 retrive with input accession key .......................................................................................................................................... 11 10.1.2 Retrieve with alternative accession key ............................................................................................................................ 12

11 Enrichment .................................................................................................................................................................................................. 12 11.1 Summarize domains ....................................................................................................................................................................... 12 11.2 Identify over-represented GO BP terms ....................................................................................................................................... 13

12 Pathway Analysis ....................................................................................................................................................................................... 14 13 Reload session ........................................................................................................................................................................................... 14

GProX Tutorial

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PREFACE

This tutorial will introduce most of the basic functionalities in GProX step-by-step. Completing the tutorial will take

approximately 1 hr. If you wish to stop at any point in the tutorial save you session and exit the program. Refer to

section 13 for instructions on how to reload the session. For a detailed description of the analysis parameters

please refer to the help file.

If you find anything not performing as expected or experience troubles going through the tutorial we would like to

know. You may post you feed back in the GProX Google Group (http://groups.google.com/group/gprox) or

contact us directly at the address listed under contacts on the project website (http://gprox.sourceforge.net)

Before starting the tutorial please ensure that you:

o Are running Windows XP or later.

o Are connected to the internet.

o Have administrator privileges on the computer.

o Have an installation of Adobe Reader

o Have .NET 3.5 or later installed. If you have older versions of .NET or are unsure go to

http://msdn.microsoft.com/netframework/ to install the most recent version.

Throughout the tutorial references to dialog parameters and input fields are indicated with this font.

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1 DATA

This tutorial goes through an example analysis of data published by Hammond et al. (Hammond, Journal of

Proteome Research, 2010). This data as a tab delimited file can be downloaded from the GProX website under

downloads and Example Data (http://gprox.sourceforge.net). Start by downloading file to your computer.

2 INSTALLATION

2.1 INSTALL REQUIRED COMPONENTS

If you are running GProX on Windows 7 you must run the program as an administrator.

If you are not running the program as administrator then exit the program and right-

click the GProX icon and select ‘Run as administrator’. If you encounter problems with

installation of components refer to the section ‘Manual Installation of Components’ in

the help file.

2.1.1 INSTALL R

1. When you launch GProX the first time you will be met with the Retrieve components dialog. If the retrieve

components dialog is not open select Get R+add-ins in the Home ribbon tab

2. Read the text in the dialog.

3. Click Start.

4. In step 1 select where you wish to install R.

5. Click Install. Download and Installation will start, depending on your internet connection this might take a

moment.

6. When the installation of R is done you will get a message and the dialog will proceed to step 2.

7. In step 2 click Install to install required R packages.

8. Once the installation is finished you will be notified and the welcome screen will be displayed

3 DATA IMPORT

3.1.1 STEP 1

1. From the welcome screen select Start new session, this will open the

input wizard.

2. Specify a Title for your session, e.g. Test session. All files in your session

will be placed in a folder with the specified title.

3. Select the input file Hammond_et_al.txt. The exact location of the file

depends on where you saved it during download.

4. The example file is tab delimited so the file format selection should be

tab-delimited.

5. Under Select Output Directory you may select in which location on your

computer the session should be created. If the Input file folder is

selected a folder with the name of your session is placed in this folder.

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6. Click Next to go to the column specification step.

3.1.2 STEP 2

1. You should see the column header in the input table listed in the left

box.

2. Move the column Protein IDs to the Accession Keys box. This column

contains the protein identifiers for the protein entries.

3. Move the following columns to the Quantitation Data box and use the

Up and Down buttons to put them in the order they are listed below:

Ratio M L Normalized 5 min

Ratio M L Normalized 30 min

Ratio H L Normalized 5 min

Ratio M L Normalized 30 min

4. Move the columns Reverse, Contaminants and Protein Names to the

Additional Data box, the order is irrelevant.

5. Click Next to proceed to Step 3 of the Wizard.

3.1.3 STEP 3

1. Check the Group ratios into experiments check box to enable the Define

Experiments panel.

2. In the Title field write EGF and select the columns:

Ratio M L Normalized 5 min

Ratio M L Normalized 30 min

and move these to the box at the right.

3. Click the Add to list button. A line should appear in the Experiment List

with the title (EGF), Color key (blue) and Columns.

4. Exchange EGF with HGF in the Title field and select the remaining two

columns and move these to the right-side box.

5. Click Add to list. You should now have two rows in the Experiment List

6. Click Next.

3.1.4 STEP 4

1. In step 4 your will see the parameters summarized.

2. Click Finish to import the data with the selected parameters.

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4 OUTPUT

4.1 SELECT GRAPHICAL FORMATS

In GProX graphical output can be created in six different formats (eps, pdf, png, mp, jpg, tif), which of these should

be created is selected in the Graphical output formats panel in the Home ribbon tab.

The formats currently selected are shown in the ribbon, to add formats expand the list and click the desired

format. To deselect a format click the format icon again, it will now be removed from the list in the ribbon.

To allow graphics to be shown inside GProX it is mandatory to create graphics in pdf and png format.

4.2 LOCATE OUTPUT

Click Browse folder in the Session panel in the Home ribbon tab. This will open your session folder where all output

will be saved in the same organization as displayed inside the program.

NB. Modifying the data tables in the session folder will lead to errors. Instead copy the file and edit the copy.

5 DATA BROWSING

5.1 COLUMNS

5.1.1 RENAME

1. Go to the Table ribbon tab.

2. Click Rename in the Columns panel.

3. Select the column Ratio M L Normalized 5 min from the list.

4. Write EGF 5 min in the New name text field and click the check mark.

5. Rename the remaining three ratio columns as below:

Ratio M L Normalized 30 min -> EGF 30 min

Ratio H L Normalized 5 min -> HGF 5 min

Ratio H L Normalized 30 min -> HGF 30 min

NB. Special characters except ‘_’are not allowed in column headers and will be replaced with white spaces.

5.1.2 MOVE

1. Select the column EGF 5 min by clicking the header cell. If you have selected

the column correctly all cells in the column should be selected.

2. Click the header cell and keep the mouse button pressed.

3. Drag the mouse to the right to move the column.

4. Move the EGF 5 min column back to its initial position as the first ratio column.

NB. The first three columns are frozen and cannot be moved.

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5.1.3 DELETE

1. Select the column Reverse.

2. Press the Ctrl keyboard key and click on the Contaminants column while keeping

the Ctrl key pressed. Both columns should now be selected.

3. Right-click somewhere in the selected area and select Columns -> Delete.

4. Click Ok in the dialog box.

NB. The first three columns cannot be deleted

5.1.4 INSERT

1. Right-click somewhere in the session table

2. Select Columns -> Insert -> From input file.

3. In the dialog select the column Peptide Counts all.

4. Move it to the right box and click the check mark.

5. The information in the selected columns for the entries is now

appended to the session table

6. Repeat steps 1-4 to re-import the columns Reverse and

Contaminants

5.2 ROWS

5.2.1 FIND

1. Select the column Reverse.

2. In the Find text field located in Table type a + symbol.

3. Click the Find button.

4. In the bottom of the data table window the number of rows containing

the search text is written and all rows containing the search string are selected.

NB. The find function is not case-specific.

5.2.2 DELETE

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To delete the reverse entries right-click somewhere in the selected area and select

Rows -> Delete.

5.2.3 MOVE

1. Select the Contaminants column and search for + as

described in section 4.1.1.2.

2. Right-click somewhere in the selected area and select Rows -

> Move to new table, this will open the New Table dialog.

3. In the dialog check the Remove selected… checkbox.

4. Select Create new table and give the title Contaminants for

the new table.

5. Click the check mark symbol . You have now created a new

Session table containing the contaminant identifications. You

can select any individual session tables by clicking the tab.

5.2.4 SORT

1. Select the Primary Accession Key column

2. Click one of the sort buttons in the menu in Sort panel in the Table ribbon tab main

window to sort the table ascending or descending.

5.3 DATA TABLE SHEETS

5.3.1 RENAME

1. Select the Session Table 1 tab.

2. Right-click somewhere in the data table.

3. Select Table -> Rename.

4. Write Data Filtered and hit enter to rename the table.

5.3.2 CLOSE

1. Select the Contaminants tab.

2. Right-click somewhere in the data table.

3. Select Table -> Close.

4. Click Ok in the dialog to close the table.

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5.3.3 OPEN

5. Right-click somewhere in the data table in the Data

Filtered tab.

6. Select Table -> Open -> Contaminants.

7. Click Open and select Contaminants.

5.3.4 DELETE

8. Select the Contaminants tab.

9. Right-click somewhere in the data table.

10. Select Table -> Delete.

11. Click Ok in the dialog to delete the contaminants table

5.4 BROWSE FUNCTIONS

5.4.1 PLOT PROTEINS

1. Select the Protein Names column.

2. Type Erk in the find field and click the Find button.

3. Right-click in one of the selected rows and select Plot

protein (s) - > Proteins together

4. Select the Data Tables window and type Grb2 in the find

field and click the Find button.

5. Right-click in one of the selected rows and select Plot protein (s) - > Experiments together

5.4.2 RETRIEVE DATABASE INFO

1. Select the Data Tables window

2. Select the Protein Names column.

3. Type EGFR in the find field and click the Find button..

4. Right-click in one of the selected rows and select Get database

entries - > IPI

5. Right-click in one of the selected rows and select Get database

entries - > UniProt

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6 NAVIGATING GRAPHICS

From the Graphics window all graphical objects are displayed and can be browsed.

6.1.1 SELECT OBJECT TO DISPLAY

From the navigation panel to the left objects can be selected. Try to display the graphics you have selected so far.

6.1.2 DELETE

1. Select the file 892_849_413_99_Proteins Together.png

2. Right-click on the filename and select Delete and click Ok in

the dialog.

6.1.3 RENAME

1. Select the file 1163_818_465_ExperimentsTogether.png

2. Right-click on the filename and select Rename

3. Write Grb2 and hit enter

6.1.4 NEW FOLDER

1. Select any file or folder in the navigation panel

2. Right-slick and select New Folder

3. Write a name e.g. Test and hit enter

6.1.5 MOVE

1. Select the file Grb2.png.

2. Drag the file into the Test folder you created in the previous section.

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7 TOOLS

7.1 TRANSFORMATION

7.1.1 LOG2 TRANSFORMATION

1. Select the Data Tables window

2. Select the Tools ribbon tab

3. In the Transformation panel ensure that the All experiment columns

list item is selected and the Log2 item is selected.

4. Click the Run button.

5. Click Yes in the dialog.

8 PLOT DATA DISTRIBUTIONS

8.1 BOX PLOT

1. Select the columns EGF 5 min and HGF 5 min

2. Right-click somewhere in the marked area and select Boxplot

8.2 SCATTER PLOT

1. Select the columns EGF 5 min and HGF 5 min

2. Right-click somewhere in the marked area and select Scatter

8.3 HISTOGRAM

1. In the Plot ribbon tab in the Plot distributions panel click the Histogram button.

2. Write EGF in the plot title field

3. Select bin size of 1.00

4. Select Juxtaposed

5. Under colors select Use experiment colors

6. In the columns field select EGF 5 min and click Add to list.

7. Under colors select specify.

8. Click the colored square next to the Fill label.

9. Select any color in the color dialog.

10. In the columns field select EGF 30 min and click Add to list.

11. Click the check mark symbol.

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8.4 HEATMAP

1. Select all four ratio columns.

2. Right-click somewhere in the marked area and select Heatmap

9 CLUSTERING

1. Select the Clustering ribbon tab.

2. Select EGF as the experiment in the Basic panel .

3. Select 6 as the number of clusters in the Basic panel .

4. In the regulation threshold panel select Fixed value and put 0.58 in the upper limit field and -0.58 in the lower

limit field.

5. Click the Run button.

10 RETRIEVE DATABASE INFORMATION

10.1.1 RETRIVE WITH INPUT ACCESSION KEY

1. Select the Query databases ribbon tab.

2. Click Select feature in the Basic panel

3. Select IPI as the query database.

4. In the feature list select UniProt

5. Click the check mark symbol.

6. In the ribbon Check the Map for secondary acc. keys.

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7. Check the Filter secondary acc. keys

8. In the Peptide column field select the Peptide Counts all column you inserted in section 5.1.4

9. Select 80 in the Threshold value field

10. Click the Run button. Depending on your internet connection the retrieval might take a couple minutes.

10.1.2 RETRIEVE WITH ALTERNATIVE ACCESSION KEY

1. In the Query databases ribbon tab check the Match with alternative keys box.

2. Select the UniProt_IPI column you retrieve in the preceding section.

3. Click Select feature in the Basic panel

4. Select UniProt as the query database.

5. Select GO, BP and Pfam in the feature list and move these to the box on the right.

6. Click the Run button. Depending on your internet connection the retrieval might take a several minutes.

11 ENRICHMENT

11.1 SUMMARIZE DOMAINS

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1. Select the Enrichment test ribbon tab.

2. In the Feature column field select the column Pfam_UniProt

3. In the p-value threshold field put 1.000

4. In the minimum number of occurrences field select 2.

5. Under Data Grouping select Clustering and select the column Cluster_EGF

6. Select 0 as the Background Cluster.

7. Click the check mark.

8. Click the Run button.

11.2 IDENTIFY OVER-REPRESENTED GO BP TERMS

1. Select the Enrichment test ribbon tab

2. In the Feature column field select the column GO_BP_UniProt

3. Select Binomial as Statistical test.

4. In the P-value threshold field put 0.05

5. In the Minimum number of occurrences field select 2.

6. Under Data Grouping select clustering and select the column Cluster_EGF

7. Select 0 as the Background Cluster.

8. Under Term filtering (GO term specific) select 2 as the Lowest hierarchical rank.

9. Click the Run button.

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12 PATHWAY ANALYSIS

1. Select the Pathway analysis ribbon tab

2. Click Select from list in the Pathway

selection panel

3. In the Select pathway dialog select the

ErbB signaling pathway (see figure to

the right) and click the check mark.

4. Under Coloring select Color by cluster.

5. Select the column Cluster_EGF as

Column.

6. Click Run.

13 RELOAD SESSION

1. Exit the program

2. Click Yes in the Save session dialog

3. Reopen GProX

4. From the welcome screen select Load Session

5. Navigate to your session folder and select the file .gpx. The name of the file is the name you gave you session

during data import.

6. Click Ok. Your session is now loaded and you can continue the analysis.