ImageMaster 2D Platinum 6.0

ImageMaster 2D Platinum 6.0
GE Healthcare
ImageMaster 2D Platinum 6.0
Tutorial
Contents
Tutorial Help
0.1
0.2
0.3
1
Experiment setup
1.1
1.2
1.3
1.4
1.5
2
Introduction ......................................................................................................13
Open an existing workspace ....................................................................13
Display gels in a worksheet .......................................................................13
Handle gels ........................................................................................................15
Organize gels in panes ................................................................................16
Adjust contrast ................................................................................................18
Profile ...................................................................................................................20
3D view ................................................................................................................22
Cursor information ........................................................................................25
Spot detection and gel matching
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
4
Introduction ........................................................................................................ 3
Display the workspace window ................................................................ 3
Create a new workspace .............................................................................. 6
Add new project ................................................................................................ 7
Import gels into your project ...................................................................... 9
Viewing and manipulating gels
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
Introduction ........................................................................................................ 1
Structure ............................................................................................................... 1
Starting a tutorial ............................................................................................. 2
Introduction ......................................................................................................27
Open an existing workspace ....................................................................27
Open gels to work on ....................................................................................27
Displaying spots .............................................................................................28
Spot detection ..................................................................................................28
Selecting spots ................................................................................................31
Editing spots .....................................................................................................31
Spot report .........................................................................................................32
Creating match sets ......................................................................................34
Matching gels ...................................................................................................37
Data analysis
4.1
4.2
4.3
4.4
4.5
Introduction ......................................................................................................43
Open an existing workspace ....................................................................43
Create classes ..................................................................................................44
Inter-Class Report ..........................................................................................48
Pick list .................................................................................................................60
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Contents
5
DIGE analysis
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
5.10
5.11
5.12
iv
Introduction ...................................................................................................... 65
Create a new workspace and project .................................................. 66
Import DIGE gels ............................................................................................ 67
Co-detecting DIGE gels ............................................................................... 69
Displaying a DIGE histogram ................................................................... 70
Displaying a DIGE report ............................................................................ 72
Creating DIGE match sets .......................................................................... 73
Matching DIGE gels ....................................................................................... 75
Creating classes with DIGE images ....................................................... 77
Comparing classes with DIGE images ................................................. 79
Match with a preparative gel ................................................................... 85
Pick list ................................................................................................................ 87
ImageMaster 2D Platinum Tutorial Edition AA
Tutorial Help
0.1
Introduction
The ImageMaster 2D Platinum tutorials offer a step-by-step guide to analyzing
your gel images and reporting results. Once you are familiar with the basic
workflow, you can refer to the user manual for instructions on how to use
additional tools and adapt procedures to your specific needs.
These tutorials are provided with gel images and related files, enabling you to
perform an entire gel analysis while highlighting the main features of the
program. Of course, you can immediately carry out the analysis on your own gel
files.
0.2
Structure
The following steps are illustrated with dedicated tutorials:
•
Tutorial 1: Experiment setup
Find out how to create a workspace to organize your experiment and how to
import gels in projects.
•
Tutorial 2: Viewing and manipulating gels
Learn how to manipulate gel images (select, move, zoom, stack) and view the
signal intensity (adjust contrast, profile, 3D view).
•
Tutorial 3: Spot detection and gel matching
Become skilled at performing automatic spot detection and matching on
your gel images (find the corresponding proteins in different gels).
•
Tutorial 4: Data analysis
Discover a powerful method to quickly find significant protein expression
variations between two populations of gels, and learn how to export a spotpicking list.
•
Tutorial 5: DIGE analysis
Learn how to carry out a complete analysis of DIGE gels using an internal
standard (importing DIGE images, observing variations within DIGE gels,
matching different DIGE gels, and finally, analyzing the protein expression
variations).
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0.3
Starting a tutorial
When you choose a particular tutorial from the Help menu, the software will close
the previously opened images and open the appropriate tutorial workspace and
files for the exercise. Each time you do this, the software restores the required files
from an archive that cannot be overwritten, and saves them in a particular folder.
In this way, you can never destroy the original tutorial files. For each tutorial, you
can restore the following states (Help > Tutorials > Tutorial X > Option ):
2
•
Restore: Closes any open files and then restores the recommended files to
start the tutorial with. The tutorial instructions are displayed in a browser
window that opens up at the right side of your screen.
•
Open: If you already worked on the tutorial gels, saved the changes and
closed the images (to look at another tutorial or process your own image
files), this opens the tutorial files with your prior modifications so that you
can continue working with the gels and workspace. The tutorial instructions
are displayed in a browser window that opens up at the right side of your
screen. If the corresponding tutorial was never loaded before, this option
automatically restores the initial state of the tutorial.
•
Results: Opens the workspace with processed files that can be expected
once all the instructions in the corresponding tutorial are run. Please note
that this overwrites your own work on the tutorial files. The tutorial
guidelines are displayed in a browser window that opens up at the right side
of your screen.
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Experiment setup
1
Experiment setup
1.1
Introduction
The workspace is the command center of ImageMaster 2D Platinum. It can be
seen as the place where all gel, matching and analysis data is centralized, and
from where all operations carried out in the software are controlled. The
workspace is a systematic and efficient way to clarify your analysis and avoid
unnecessary work. Ideally, it should reflect the structure and design of your
research. It is necessary to setup a workspace to analyze new gels.
Note that this tutorial in particular describes the processing of non-DIGE gels.
Most of the procedures and functionalities will also apply to DIGE gels. Please
refer to Tutorial 5 to learn about the specificities of a DIGE gel analysis.
Tutorial gels
When you select Help > Tutorials > Tutorial 1 > Restore in the menu, any open
worksheets and workspace are closed and the Workspace window containing an
empty workspace is opened. In this tutorial, you can import the 6 TIFF files that
can be found in C:\Program Files\GE Healthcare\ImageMaster 2D
Platinum\Tutorials\Tutorial1 (see directives below).
1.2
Display the workspace window
The workspace is displayed in a dockable window, like tabular reports and
graphical reports. Dockable windows enable you to manage the many
documents that may need to be open while doing your analysis. But whereas
reports can be moved in different positions and closed at any time, the
Workspace window is always displayed at the left of the Gel Display Zone, and
cannot be closed due to its crucial role. It can be in Pinned, Auto-Hide or Floating
mode.
1
Click the Workspace tab below the ImageMaster toolbar to display the
Workspace window.
By default, the Workspace window is displayed in Auto-Hide (Un-pinned) mode.
It will automatically hide or collapse when not in use, to form a tab alongside the
edge to which it is docked.
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Experiment setup
2
You can click on the Collapse icon to hide the Workspace window
3
Click again on the Workspace tab to display the Workspace window.
To display the Workspace window in Pinned mode:
4
4
Click on the Pin icon at the top-right corner of the window. The icon is
changed to indicate that the window is now in Pinned mode. In this
mode, the window is locked and therefore will not be hidden when not
used. You can move a Pinned window by dragging its title bar.
ImageMaster displays blue arrow guides to indicate where the window
may be locked. In the case of the workspace, only the left position is
allowed.
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To display the Workspace window in Floating mode:
5
Drag the window title bar outside the blue arrow guides. In this mode,
the Workspace window can be placed anywhere on your screen (even
outside the boundaries of the main program window). Note that the
Close icon is unavailable in the case of the Workspace window, but it can
be used to close reports.
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Experiment setup
To revert to the Pinned mode:
6
1.3
Drag the window title bar in one of the blue guides. The window is now
in Pinned mode.
Create a new workspace
To create a new workspace:
1
6
Click the Workspace tab below the ImageMaster toolbar to display the
Workspace window:
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Experiment setup
2
Click on the New icon
3
Specify a workspace name and a destination folder for the new
workspace file (.mws). You can also enter a comment to help you
remember later on what you did and why. If you work on the tutorial
gels, enter Tutorial1 in the Workspace Name field, and browse the
folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial1 to save your
file. Click OK when you are finished.
1.4
in the workspace toolbar.
Add new project
In the Workspace window, you will find that your new workspace does not
contain a project. A project includes all gels, along with related data, produced
and analyzed during the course of a specific gel study.
To add a new project to your workspace:
1
Right click on the workspace name (Tutorial1) at the top of the
workspace navigator.
2
Choose the option New Project from the contextual menu.
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Experiment setup
3
Enter a new project name, a destination folder for the project file (.prj)
and a comment that describes the project. For the purpose of this
tutorial, use the name Bacteria, the same folder as before, and enter a
comment as indicated below. Click OK.
You find that your workspace now includes a project called Bacteria, containing
five default folders: Gels, MatchSets, Classes, Reports and Documents. Please note
that if you purchased a license for ImageMaster 2D Platinum DIGE, a subfolder
called DIGE Gels exists in the Gels folder. See chapter 5 of these tutorials to learn
how to use this DIGE Gels folder.
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Experiment setup
1.5
Import gels into your project
Now, gel images must be incorporated into the Gels folder of your project.
1
Right click on the Gels folder in the workspace navigator. Choose the
option Import Gels in the contextual menu.
2
In the Import Image window, select the input format of the files and
indicate the reduction factor (for example, 2 to reduce the resolution
from 300 to 150 dpi). For the provided tutorial gels, the reduction factor
should be 1 and the file format TIFF. Then, click OK.
3
Browse the folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial1 and
select the six TIFF images provided with this tutorial, while holding the
Ctrl key. Click Open to import the gels.
4
The imported images are saved with the extension .mel. You can change
the file names manually, automatically add an extension to all the
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Experiment setup
existing file names, or decide to save the files in a different folder. For the
purpose of this tutorial, just click OK to save the .mel files in the proposed
folder.
5
Then, enter the staining method that has been used to run this gel, by
choosing the appropriate one from the proposed list.
The imported gel files now appear in the Gels folder of the project. When you
expand the Gels folder, your workspace should look like the figure below.
The added gels can be grouped in subfolders within the Gels folder of the project.
6
10
Right-click on the Gels folder, and choose the Create Folder option. Enter
AT1 as a name for the new folder.
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Experiment setup
7
Select the gels A_T1_Gel1, A_T1_Gel2 and A_T1_Gel3, while holding the
Ctrl key, and drag and drop them on the new AT1 folder.
8
The subfolder AT1 now contains the three gels. Repeat the procedure to
create a subfolder called AT2, which contains the three other gels.
Save the changes made to the project by clicking on the Save icon
in the
workspace toolbar. This does not save any changes made to your gels: gels and
workspace are independent, and therefore need to be saved separately!
Please note that you can create as many projects as you want in your
workspace. You may also add existing projects: right click on the workspace
name, choose Insert Project in the contextual menu, browse the folder where the
project file is located, and select it (file with a prj extension).
Later, when gel matching must be performed, match sets are created within the
workspace (see Tutorial 3), then classes are defined to carry out statistical
analysis (see Tutorial 4).
If you select gels in the workspace, you will see that detailed information about
selected files is given on the right side of the Workspace window. The details
include the Name, MatchSet and Class to which the image belongs, dates
Created and last Modified, Size (in bytes), File Type, File Path and ID.
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Experiment setup
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Viewing and manipulating gels
2
Viewing and manipulating gels
2.1
Introduction
ImageMaster 2D Platinum offers many functionalities and tools to manipulate
and view gels and their intensity profiles. This tutorial familiarizes you with some
of these features.
Tutorial gels
For this tutorial, you can continue working with the workspace created in Tutorial
1. Alternatively, you can select Help > Tutorials > Tutorial 2 > Restore in the menu.
This closes any open files and displays a workspace called Tutorial2 in the
Workspace window.
2.2
Open an existing workspace
In case you work on your own image files, first open a workspace containing your
gels, created using the instructions in Tutorial 1:
1
Click on the Workspace tab to display the Workspace window.
2
Click on the Open icon
workspace.
3
Browse the appropriate folder and open your workspace file (.mws).
2.3
in the toolbar of this window to open a
Display gels in a worksheet
Gel images are always opened in Worksheets. Each worksheet has a banner
containing its name and type (enclosed in brackets []). The worksheet type
corresponds to the name of the workspace folder from where the gels are
extracted (Gels, MatchSets or Classes). Please note that you cannot open gels
from different folder types in a same worksheet. Within a worksheet, gel images
are grouped in Panes according to their belonging to folders or sub-folders in the
project.
To open gels in a worksheet:
1
Select the subfolders AT1 and AT2 in the Gels folder, by clicking on their
names while holding down the Ctrl key.
2
Right click on one of the subfolder and choose Open > In Worksheet in
the contextual menu. You can see that the six gels are displayed in two
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Viewing and manipulating gels
panes, one for each sub-folder: AT1 and AT2. The worksheet is named
automatically AT1-AT2.
14
3
Select the pane called AT1 by clicking on its banner. All the gels within
this pane are now selected (pane banner and gel legends in green)
whereas those from AT2 become unselected (pane banner and gel
legends in grey).
4
Close the pane AT1 by choosing File > Close Images in the menu. Only
the pane AT2 remains in the worksheet AT1-AT2.
5
Open the AT1 gels again, but in a different worksheet, by selecting the
sub-folder AT1 in the workspace, right-clicking on it, and choosing Open
> In New Worksheet. A new worksheet, called automatically AT1, is
created, and it appears at the front of the Display Zone, while the
previous worksheet is now inactive (banner in grey). Please note, that
there is always just one active worksheet.
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Viewing and manipulating gels
To change the active worksheet:
6
Switch the worksheets by clicking on the banner of AT1-AT2.
To close a worksheet:
2.4
7
Select all the gels displayed in worksheet AT1-AT2 with Select > Gels > All
in the menu.
8
Close them with File > Close Images in the menu.
Handle gels
Within a worksheet, you can manipulate a single gel image or several images at
a time.
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Viewing and manipulating gels
•
To move gels, use the Hand tool
from the toolbar, and drag the gels to
the desired location. You can also use the sliders at the right and bottom
edges of each image.
•
With the Magnify tool
, you can either zoom in (left mouse button) or
zoom out (right mouse button). You can also zoom by resizing the sliders at
the right and bottom edges of each image. If you only need to briefly
examine some details, you can use the Magnifying glass by shortly holding
the Ctrl key while pressing one of the mouse buttons and moving your
cursor over the interesting region.
•
To select only a portion of the image, activate the Region tool
, then
click and define the interesting area. The selection can be moved by clicking
inside the region and dragging, or resized by clicking on its borders or
corners. Deselect a region by clicking outside it. Defining a region can be
necessary for previewing contrast mapping or spot detection, for cropping
gels, or just for selecting objects in the region. Some uses are illustrated in
the rest of the tutorials.
•
When holding the Shift key while moving or zooming gels, or defining gel
regions, the manipulations are carried out on all gels (even deselected gels).
•
After moving or zooming a particular gel, double clicking with the Hand tool
in this gel moves all the other gels (even deselected ones) to the same
location, and with the same zoom factor.
•
To select gels to work on, you can activate several images by holding down
the Ctrl key as you click on their legends. You can also select several gels by
clicking on the first gel legend and then, while holding down the Shift key,
clicking on the legend of the last gel in a desired series. All gels in between
become selected.
•
To select all the gels in the active worksheet, choose Select > Gels > All in the
menu or use the Ctrl+A keyboard shortcut.
2.5
Organize gels in panes
You can stack gels within a pane, putting them one on top of the other. This keeps
a reasonable display size for each gel when working with many gels. To stack
images and manipulate image stacks:
•
16
Make sure the pane is selected by clicking on its banner if necessary.
Choose View > Pane Layout > Stacked in the menu. All the gels of the pane
are now stacked.
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Viewing and manipulating gels
•
A hidden gel in the stack can be made visible by clicking on its tab at the
bottom of the pane.
•
To switch between stacked gels, you can use the Page Up or Page Down
keys on the keyboard.
•
Double click with the Hand tool on a gel region to re-center all the gels on
the same position.
•
To select all gels in a stack, click on the pane banner.
•
You can use View > Pane Layout > Free in the menu, in order to define the
number and the organization of cells (columns and rows) to display in the
selected pane. For instance, if you choose to display the pane with 1 row
and 3 columns; you will get the following configuration:
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Viewing and manipulating gels
NOTE! In the same way, panes can be stacked within a worksheet by using
the different options of View > Worksheet Layout (Stacked/Tiled/Free) in the
menu.
•
Move the legend of A_T1_Gel3 on the gel A_T1_Gel1 to invert their position
in the pane. Then, choose View > Switch (or Ctrl+F) to cancel the previous
operation and therefore to allow a quick switch between the two images.
•
You can return to the original image organization by selecting View > Pane
Layout > Tiled in the menu.
2.6
Adjust contrast
Play with the contrast settings to get a feeling for your image quality:
1
18
Select one or more gels.
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Viewing and manipulating gels
2
Draw a preview region in one or more (use the Shift key) gels with the
Region tool.
3
Choose Show > Gels > Adjust Contrast in the menu. It opens the Image
Display Settings window.
4
By moving the left or right borders of the slider below the histogram (for
the gel indicated in the Image field) or by changing the Bending
parameter, you can modify the contrast and brightness of the image,
and see the effect on the selected regions.
5
Checking the Only In Region box allows you to display the gray level
histogram for the preview region only.
6
Use the desired color option at the top of the window to modify the color
palette. Different predefined palettes are available. Please note that
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Viewing and manipulating gels
whatever palette is chosen, it can only be applied to all or none of the
gels (even those that are not selected or in a selected worksheet).
7
At any time (while the Image Display Settings window is open) you can
select other gels or redraw the preview regions, to see what the effect of
the contrast mapping would be on the selected gels.
8
Click OK to apply the contrast adjustments to the selected gels.
Please note that while contrast mapping changes are saved with the gel image
(without changing the raw data), color palettes are not saved in the gel file.
2.7
Profile
The Profile function gives a horizontal and vertical section of the gel at the
position of the mouse cursor, thus showing the intensity variation in the X and Y
20
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Viewing and manipulating gels
directions. To display the profile, ImageMaster automatically loads the raw image
data of the gels in the current worksheet.
1
Choose Show > Gels > Profile in the menu.
2
Hold your mouse cursor over the desired gel to view the profiles.
3
To deactivate the Profile feature, untick Show > Gels > Profile in the
menu.
4
You can unload the raw image data to regain memory. To do so, select
Edit > Gels > Raw Image > Unload in the menu. This is only necessary
when your memory resources are low.
Before going on with the following section, reorganize your gels once again:
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Viewing and manipulating gels
5
2.8
Choose View > Pane Layout > Free in the menu, and choose 3 rows and
1 column.
3D view
Another way to examine the intensity variations in a gel is by looking at the threedimensional (3D) view of a gel region. In such a view, the isoelectric point (pI) and
molecular weight (MW) values are, as usual, represented by the X and Y axes,
whereas the pixel intensity is plotted along the third dimension (Z axis).
The resulting image shows a peak for each protein spot, with a peak height that
is proportional to the spot intensity.
To display a 3D view of a single gel region:
22
1
Select the gel for which you would like to display the 3D view.
2
Select a region in this gel. Alternatively, you can select one or more spots
using the Spot tool.
3
Choose Reports > 3D View in the menu.
4
When spots are selected and moreover a region is defined, the software
asks whether your 3D view should be based on the selected region or
selected spots.
5
ImageMaster displays a 3D view for the active region or the area around
the selected spots.
6
Pin the dockable 3D View window by clicking on the
3D View beside the gels.
icon to show the
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Viewing and manipulating gels
7
By clicking on the arrows, at the right side of the 3D view, you can turn
the 3D view in all directions or zoom in or out.
8
Right click anywhere in the 3D view to center the view on the clicked
position.
9
You can hide the axes in the view by selecting the Display Axes option
under the Show icon in the 3D View window. Repeat the operation to
display the axes again.
The 3D View feature in ImageMaster enables comparative viewing of
corresponding regions or spots in different gels. To display multiple 3D views of
several (matching) gel regions:
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Viewing and manipulating gels
10 Use Shift to select the same region in all open gels, using the Region tool.
11 Select the gels for which you would like to display and compare the 3D
views (select the three AT1 tutorial gels with the Ctrl+A shortcut).
12 Choose Reports > 3D View in the menu.
13 ImageMaster displays a new 3D View window on top of the previous
one. It displays the 3D views of the active regions side by side.
14 Use the arrows at the right side of the window to simultaneously rotate
all the gel regions. Note that the different views are displayed with the
same scale. Therefore, you can easily visualize any protein expression
changes.
15 If your 3D views are centered on spots, the heights of the different views
may differ. If you right click on the background in one of the views, while
holding the Shift key, you will center all the views on this new position. If
this position also corresponds to background in the other gels, the views
will now be at the same height.
24
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Viewing and manipulating gels
16 You can visualize again the first 3D View window (that displayed the
single gel region). To do so, click on its tab at the bottom of the docked
window.
17 Close the 3D view windows.
2.9
Cursor information
To view information about pixels and spots (once spots have been detected), the
Cursor Information window can be displayed:
1
Choose Window > Cursor Information in the menu.
2
The Cursor Information window appears on the screen.
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Viewing and manipulating gels
26
3
Click on the Settings icon to choose the information to be displayed.
4
In the pop-up dialog box, select attributes in the Visible list and click on
the blue left-pointing arrow to hide them, and vice versa to show any
Hidden attributes. Click OK to confirm your choice. These settings can be
made permanent for a particular workspace by going to the Cursor
Information tab in the ImageMaster Options (choose Tools > Options in
the menu).
5
Hold your cursor over the pixel of interest. The Cursor Information
window displays the desired information. If the Value field shows
Unloaded, the raw data are not available. In this case, choose Edit > Gels
> Raw Image > Load.
6
Close the Cursor Information window.
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Spot detection and gel matching
3
Spot detection and gel matching
3.1
Introduction
Spot detection in ImageMaster 2D Platinum was optimized to yield relevant
biological results with minimum user interaction. Only a few straightforward
parameters need to be set to automatically locate the spots in a gel image. Once
this is done, the gels can be matched, possibly using one or two landmarks.
Note that this tutorial in particular describes the processing of non-DIGE gels.
Most of the procedures and functionalities will also apply to DIGE gels. Exceptions
are the spot detection parameters and quantification values. Please refer to
Tutorial 5 to learn about the specificities of a DIGE gel analysis.
Tutorial gels
For this tutorial, you can continue working with the workspace created in Tutorial
1 and used in Tutorial 2. Alternatively, you can select Help > Tutorials > Tutorial 3
> Restore in the menu. This closes any open files and displays the Workspace
window with a workspace called Tutorial3.
3.2
Open an existing workspace
In case you work on your own image files, first open a workspace containing gels,
created using the instructions in Tutorial 1:
1
Display the Workspace window.
2
Click on the Open icon
workspace.
3
Browse the appropriate folder and open your workspace file (.mws).
3.3
in the toolbar of this window to open a
Open gels to work on
Open the gels to work on during this session:
1
Select the subfolders A_T1 and A_T2, by clicking on their names while
holding down the Ctrl key.
2
Right click on one of the names and choose Open > In Worksheet in the
contextual menu.
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Spot detection and gel matching
3.4
Displaying spots
ImageMaster allows you to display spots in different ways. Before starting spot
detection, it is best to change the spot shape to the Outlined mode:
Crossed
1
Select all the gels in the worksheet (Ctrl+A).
2
Choose Show > Spots > Shape > Outlined in the menu.
Outlined
Filled
Outlined/Filled
Note that the default mode can be changed for a particular workspace by going
to the Display tab in the ImageMaster Options (choose Tools > Options in the
menu).
3.5
Spot detection
To detect spots efficiently, we recommend previewing the spot detection results
on a few small gel regions (drawn with the Region tool before or during spot
parameter optimization) so that you can decide whether the parameters need to
be fine-tuned or not. Each change in one of the spot detection parameters is
immediately reflected in the selected region. Once you find the optimal
parameters, you can detect spots on all selected gels.
To detect spots automatically:
28
1
Select all gels (Ctrl+A).
2
Draw a region with the Region tool on one or more selected gels in areas
with representative spots.
3
Choose Edit > Spots > Detect in the menu. The Detect Spots window
appears on the screen, and the spots in the active regions are detected
with the default parameters.
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Spot detection and gel matching
4
Adjust the Smooth parameter to detect all real spots and correctly split
any overlapping ones (a value of 2 works well with the tutorial gels). At
this level, you should not be concerned with the high number of noise
spots. These are filtered out with the two other parameters.
5
Choose Window > Cursor Information in the menu. Move your cursor
over some spots that you consider to be noise or artifacts. Look at the
Saliency values for these spots.
6
Enter a Saliency value in the spot detection window that is just above
that of the spots to be filtered out. All spots with Saliencies lower than
the given threshold will be removed (use a Saliency cutoff of 150 with the
tutorial gels).
7
In some gels, very small but intense artifacts (for example dust particles)
remain detected and cannot be eliminated with the Saliency parameter
without removing real spots. Such artifact spots can be removed by
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setting an appropriate Min Area value (expressed in number of pixels).
With the tutorial gels, it is not necessary to use this parameter.
8
When the detection preview is satisfactory, click on OK to detect all
spots in the selected gels using the parameter values that were set.
9
The spot shapes are displayed on the gels.
10 Choose File > Save > Worksheet to save the spot detection results.
11 Select the Region tool and double click to make the preview region
disappear. If holding the Shift key while doing this, the preview regions
from all gels are removed.
12 Close the Cursor Information window.
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3.6
Selecting spots
Spots can be selected with the Spot tool as long as they are visible on the gel.
Once selected, the spots are highlighted in green.
•
To select a spot, make sure the Spot tool
is activated and then click on
the spot. To select more than one spot, select the first one, and then hold
the Shift key down while clicking on additional spots.
•
To select all spots in a region, position the cursor at the top left position of
the desired region, hold down the mouse button, and then drag the cursor
to the bottom right position.
•
To deselect all spots, select the gels for which you would like to deactivate
the spots, and click in one gel (not on a spot).
•
You can also select spots by using the options in the Select > Spots menu.
3.7
Editing spots
Quantitative protein data, and in particular the spot volume, are highly
dependant on an optimal and reproducible definition of the spot borders and a
correct splitting of partially overlapped spots. To increase the precision of
quantitative work, it is therefore highly recommended to create spots using the
automatic spot detection algorithm in ImageMaster. It is not advisable to
manually edit spots because this introduces significant quantification errors.
Please refer to the ImageMaster user manual for instructions if you still prefer to
edit spots yourself (split, merge, grow and reduce).
You can of course delete spots:
1
Select the spots to be deleted.
2
Choose Edit > Spots > Delete in the menu.
Tips for spot editing:
•
Make sure all real spots are detected and correctly split in the spot detection
process. If artifacts are detected, you can always filter them based on
Saliency, Min Area or, if this tends to remove also real spots, based on spot
quantification measures such as Intensity, Volume, %Intensity, %Vol, Area.
•
Instead of splitting or merging spots when two spots in one gel seem to
correspond to a single (larger) spot in another gel, you can compare the
summed quantification values of the two spots with the quantification value
of the single spot. To make such comparisons easier, ImageMaster allows
you to create and select multiple matches between one spot in the master
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gel and several spots on the other gels in the match set but not the inverse
(see the user manual for details).
3.8
Spot report
A Spot Report summarizes relevant information about the selected spots. To
display this report:
32
1
Select the gels containing the spots you are interested in.
2
Select spots with the Spot tool or menu options.
3
Choose Reports > Spot Report > Current Template. A Spot Report is
displayed.
4
Click on the Settings icon to choose the information to be displayed. In
the pop-up dialog box, select attributes in the Visible list and click on the
blue left-pointing arrow to hide them, and vice versa to show any hidden
attributes. Click OK to confirm your choice. Note that you can include
attributes from the master gel (see section below on matching).
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Several features are listed below that apply to the Spot Report, and in general to
all other ImageMaster reports:
•
A Spot Report can be saved
, printed
, or selected lines in the
report can be copied to the clipboard
for pasting in other software
such as MS Excel.
•
Spots can be selected from reports by double clicking the corresponding
line or by selecting the spot(s) in the report and clicking the Select on Gels
icon
in the report toolbar.
•
One can also select and view the Next
•
The spots can be sorted according to one of the criteria displayed in the
Spot Report, by simply clicking on the header of the corresponding column.
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spot on the gel.
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3.9
5
Close the Spot Report window.
6
Close the AT1-AT2 [Gels] worksheet by choosing File > Close All in the
menu.
Creating match sets
One of the major innovations of ImageMaster 2D Platinum 6.0 is the possibility to
match populations of gels instead of only matching gels. The population
matching requires the definition of one or more match sets, which include gels or
populations of gels that should be matched together.
In this tutorial, you will match two populations AT1 and AT2. To do so, you must
first create a match set for each population within the workspace:
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1
In the Gels folder of the Bacteria project, expand the folder AT1.
2
Select the three gels within AT1 by clicking on their names while holding
down the Ctrl key.
3
Right-click on one of the selected gels, and choose Add In MatchSet in
the contextual menu.
4
In the Add Gels In MatchSet window, enter AT1 as a name for the match
set to be created, and click OK.
5
Define a reference among the gels, which will be used to create a Master
image for the matching. In our example, choose AT1_Gel1, and click OK.
In the MatchSets folder, a match set called AT1 is now displayed, which
contains the three added gels. The gel chosen as the reference has a
specific icon (with a red component).
6
Right-click on the match set AT1, and choose Open in the contextual
menu.
7
A [MatchSet] worksheet is opened. The master gel Master_AT1 is
automatically shown (with a red legend) in this worksheet. The reference
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gel (A_T1_Gel1) only appears if you choose Show > Show Reference in
the menu (or press the F1 key).
8
Repeat the procedure (steps 1 to 7) to create and open a match set AT2,
which contains the three gels of AT2 in the Gels folder (choose
A_T2_Gel1 as a reference to create the Master).
9
Close the open [MatchSet] worksheets by choosing File > Close All in the
menu.
In the following step, you will create a higher level match set to allow the
matching of populations AT1 and AT2.
10 Right-click on the MatchSets folder of the project, and choose Create
MatchSet in the contextual menu.
11 In the Create MatchSet window, enter A as a name for the new match
set, and click OK. An empty match set, A, is created in the Match Sets
folder.
12 Drag and drop the match sets AT1 and AT2 onto A. You are asked if you
want to move them inside or after A. Answer Inside.
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13 The match sets AT1 and AT2 should now be in match set A.
14 Right-click on match set AT1 and choose Set MatchSet as Reference in
the contextual menu. The match set AT1 gets a specific icon (with a red
component and a tick mark) to show that its master image will be used
to create the master of match set A.
3.10
Matching gels
You can now effectively match , first the gels in each sub match set, then the
match sets AT1 and AT2.
First, open match set AT1:
1
In the Workspace window, right-click on the match set AT1, and choose
Open in the contextual menu. Note that, by default, the reference
A_T1_Gel1 is not visible. It doesn’t need to be displayed since it is
automatically matched with the master. You can display the reference
gel by choosing Show > Show Reference (or F1 key) in the menu.
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ImageMaster was designed to match gels with minimum user intervention, but
sometimes you may have to help the matching process by specifying a few
landmark annotations (generally only one or two).
To position a landmark:
38
2
Make sure all spots are deselected by first selecting all gels (Ctrl+A) and
then choosing Select > Unselect All in the menu.
3
Using the Shift key, select the same spot on all gels with the Spot tool
(choose small, well-defined, unambiguously corresponding spots that
are not in distorted regions).
4
Choose Edit > Annotations > Add Label in the menu.
5
Choose the category Landmark. Click OK.
6
Supply a name such as L1.
7
Click OK to close the window, and Yes to confirm that you want to add
labels to your gels.
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To perform automatic matching:
8
Select all the gels (Ctrl+A).
9
Choose Edit > Matches > Match Gels in the menu.
10 All the gels are matched to Master_AT1.
11 When matching is completed, ImageMaster gives the total number of
matches found. Click OK to close this message.
12 Choose File > Save > Worksheet to save all modifications (annotations
and matches) done in this worksheet.
To view the matching results:
13 Show the match vectors between the spots in the front gel and those in
the master by choosing Show > Matches > Show Vectors in the menu.
14 Vectors are shown for all gels but the master gel. These vectors are
automatically minimized in the displayed region.
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You can check the vector pattern for consistency. If there is a mismatch, the
vector will have a different length and/or orientation.
15 Show the spots from the master by selecting Show > Gels >
Transparency > Show in the menu. In the Transparency Settings window,
choose Master_AT1 as the transparency reference, and tick the Spots
Overlapped option, then click OK.
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16 The red spots are always those from the current gel, the blue spots those
from the specified transparency reference.
17 The blue spots are not necessarily well superimposed on the red spots.
To solve this, make sure the Hand tool is still selected, and then double
click on a particular position on the gel. All your gels now have the same
zoom factor and are centered at the same position. The distances
between the red spots and the corresponding blue spots in the
transparency reference are minimized in the clicked region.
18 Hide the spots from the master (transparency reference) by selecting
Show > Gels > Transparency > Hide in the menu.
If you observe mismatches, you can delete bad matches or manually add new
ones. To edit matches:
•
Delete bad matches by selecting the corresponding spot(s) and choosing
Edit > Matches > Delete Match in the menu.
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•
Manually add matches by selecting the relevant spots, and choosing Edit >
Matches > Add Match in the menu.
•
You can also add multiple pairs, that is, one spot in the master gel can be
matched to several spots in another gel. First select the spot in the master
gel and then select (hold the Shift key) all the spots that should be matched
with it. Choose Edit > Matches > Add Match in the menu to add matches
between all selected spots and the spot in the master gel, thus forming a
spot match. The image below, showing a corresponding region on the gels
in tiled mode, best illustrates the notion of multiple matches; a single spot in
the master gel corresponds to a double spot in A_T2_Gel2..
19 Repeat the matching procedure (steps 1 to 18) for match set AT2.
20 Then open match set A from the workspace by right-clicking on its name
and choosing Open in the contextual menu. Again, the reference gel is
not open since it is automatically matched with the master of A. You can
match Master_AT2 against Master_A, using the same procedure as for
the matching of any other gel.
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4
Data analysis
4.1
Introduction
ImageMaster 2D Platinum offers several tools for examining gel data and finding
protein expression variations. This tutorial describes one of these analysis
methods. It provides a quick way to find significant expression differences
between populations of gels.
Note that this tutorial in particular describes the processing of non-DIGE gels.
Most of the procedures and functionalities will also apply to DIGE gels. Please
refer to Tutorial 5 to learn about the specificities of a DIGE gel analysis.
Since the exercise concentrates on the analysis of protein expression changes
between populations of gels, a larger set of sample gels is used. In the following
experiment, four populations exist. Cells were grown under two different
conditions (using substrate A and substrate B) and underwent one of two
treatments (treatment 1 and treatment 2).
Class
Condition
Treatment
A_T1
Substrate A
Treatment 1
A_T2
Substrate A
Treatment 2
B_T1
Substrate B
Treatment 1
B_T2
Substrate B
Treatment 2
Tutorial gels
For this tutorial, you can continue working with the workspace created in Tutorial
1 and used in Tutorials 2 and 3. However, as mentioned above, additional gels and
match sets have been added in the Tutorial4 workspace in order to demonstrate
the power of the project organisation. Choose Help > Tutorials > Tutorial 4 >
Restore in the menu to open this extended tutorial workspace.
4.2
Open an existing workspace
In case you work on your own gel files, first open a workspace containing gels
that were detected and matched (please refer to Tutorials 1 and 3 to learn how to
create a workspace with gels and match sets, and how to carry out spot
detection and gel matching).
1
Display the Workspace window.
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2
Click on the Open icon
workspace.
3
Browse the appropriate folder and open your workspace file (.mws).
4.3
in the toolbar of this window to open a
Create classes
In order to carry out comparisons of gels or gel populations, classes have to be
defined. A class is a set of gels or gel populations with common biological
properties that you can compare with other such entities. Comparing classes will
enable you to find the protein expression variations between different biological
states.
To illustrate the use of classes in the ImageMaster project organisation, two
different match set structures have been created in the Tutorial 4 workspace. In
match set Bacteria1, the three gels from each subpopulation (AT1, AT2, BT1, BT2)
were first matched together before creating match set A from AT1 and AT2, and
B from BT1 and BT2. In match set Bacteria2, all six gels from population A were
matched in a first step, as were all six gels from population B. The resulting match
sets Abis and Bbis were then matched to give Bacteria2.
Although these match sets use the same gels (and detection), the data analysis
results may differ more or less depending on which match set is used for creating
the classes to be analyzed. In fact, any of these match set structures can be
justified and provide good results. You can even use both to confirm your results
or maybe find a few additional protein markers.
To show how this works, the classes Bacteria1 and Bacteria1b have already
been created in the Classes folder of the Bacteria project. These two classes are
based on the match set Bacteria1. Class Bacteria1 reproduces the organisation
found in the corresponding match set. A configuration as in class Bacteria1B
rather allows you to compare all gels that underwent treatment T1 with those
that were in group T2. You will now learn how to create a new class using the
same structures as in class Bacteria1 but based on the match set Bacteria2:
44
1
In the Workspace window, expand the match set Abis within Bacteria2,
and select the gels A_T1_Gel1, A_T1_Gel2 and A_T1_Gel3 by clicking on
their names while holding the Ctrl key.
2
Right-click on one of the selected gels, and choose Add In Class in the
contextual menu.
3
In the Add Gels In Class window, enter AT1bis as a name for the created
class, and click OK.
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4
A class, called AT1bis, is created in the Classes folder. Note that you can
look at the MatchSet column in the File Details (at the right side of the
workspace window) to see from which match set a gel is derived
(Bacteria2\Abis in this case). It allows you to verify that the gels belong
to the same match set, which is necessary for statistical comparisons.
5
Repeat the procedure (steps 1 to 4) to create the classes AT2bis, BT1bis
and BT2bis, always selecting the gels from the match set Bacteria2.
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46
6
Right-click on the Classes folder and choose Create Class in the
contextual menu. In the Create Class window, enter Bacteria2 as a name
for the new class.
7
Now right-click on the class Bacteria2 and choose Create Class in the
contextual menu. In the Create Class window, enter Abis as a name for
the new class. Repeat this procedure to create a class Bbis within the
class Bacteria2.
8
Select the classes AT1bis and AT2bis by clicking on their names while
using the Ctrl key, and drag and drop them in the empty class Abis.
Answer Inside when asked to move the classes Inside or After class Abis.
9
Select the classes BT1bis and BT2bis by clicking on their names while
using the Ctrl key, and drag and drop them in the empty class Bbis.
Answer Inside when asked to move the classes Inside or After class Bbis.
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10 Select AT1bis and AT2bis using the Ctrl key and right click on one of their
names. Choose Open in the contextual menu.
11 A pane is displayed for each class in the new [Classes] worksheet. It is
possible to display the master gel by choosing Show > Show Master in
the menu (or using the F1 key). The Master corresponds to the first
common node in the match set structure, that is, the master gel of
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match set Abis in the present case. Select Show > Show Master again (or
press the F1 key) to make the Master disappear.
Please note that to compare gel populations, the gels should have a common
node in the match set that is used to construct the classes. Without a common
node, the gels cannot be matched together and statistical comparison is
impossible.
4.4
Inter-Class Report
You are now ready to display an Inter-Class Report enabling you to find the
protein expression variations between the classes AT1bis and AT2bis. In the InterClass Report you can display, for every selected match, the central tendency (e.g.
mean value) and dispersion of the spot values in each class. Moreover,
ImageMaster 2D Platinum computes overlapping measures between the class
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intervals, which are defined by the range [Central value - Dispersion, Central
value + Dispersion]. The overlapping measures were implemented because the
ratio between the mean values in each class, often used by researchers to find
protein expression changes, is only a very partial measure to express the
difference between two populations. Matches characterized by a relatively high
ratio between two classes can still have a lot of spot values that overlap. This is
illustrated by the histograms below, which display the individual spot values
(orange bars) in each class (separated by a gray vertical line). The horizontal blue
line indicates the mean value for each class, and the red lines define the
dispersion intervals.
111
461
496
Normal Ratio*
-5.78514
6.71484
6.04695
Overlap Ratio*
0
0.730560
1.12634
Overlap Gap
-0.0721690
-0.00982704
0.00766187
76
466
473
Normal Ratio*
3.08088
-2.58183
-3.24276
Overlap Ratio*
1.93352
-1.89343
-2.67644
Overlap Gap
0.0983950
0.0832530
0.102301
* Negative values mean that the spots are under-expressed in the second class
when compared with the first one. Positive values indicate that the spots are overexpressed in the second class, compared to the first class.
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The groups 461, 496 and 111 have a "Normal Ratio" (ratio between the mean
values for each class) close to 6. However, their class intervals may overlap
entirely (match 111) or partially (match 461).
On the other hand, matches with lower "Normal Ratios" such as matches 76, 466
and 473 can be much more interesting. The individual values are quite
homogenous within each class and there is no overlap between the two classes.
The overlapping measures Ratio and Gap, as calculated in ImageMaster 2D
Platinum are more powerful to find protein expression changes. You can find
details and schemes in the user manual. In short, and by slightly simplifying:
Ratio (Overlap Ratio): Ratio between the lower limit of one class interval (class
with the highest mean value) and the upper limit of the other class interval (class
with the lowest mean value). Absolute values smaller than 1 indicate overlap,
whereas absolute values higher than 1 show that there is no overlap between the
class ranges. If one class interval is completely overlapped by another one, you
will find a 0 value for the Ratio. Negative values indicate that the spots in the
current class are under-expressed compared to those in the other class.
NOTE! In the rest of the tutorials, “Ratio” will designate the “Overlap Ratio”, as
calculated by ImageMaster. Please note that you can calculate the “Normal
Ratio” by setting the dispersion percentage slider in the dialog to 0% (see
below).
Gap (Overlap Gap): Difference between the lower limit of one class interval (class
with the highest mean value) and the upper limit of the other class interval (class
with the lowest mean value). Negative values indicate overlapping intervals
whereas positive values correspond to non-overlapping class ranges.
You will find that sorting your matches based on these two criteria is a very
efficient way to detect interesting protein expression changes. Proceed as
follows:
50
1
Choose Select > Gels > All (Ctrl+A) in the menu to select all gels. Only
selected gels will be included in the analysis.
2
Select all matches by choosing Select > Matches > All in the menu.
3
Choose Analyze > Inter-Class > Report in the menu.
4
Select the value type to be used. In most non-DIGE applications,
including this tutorial, using the %Vol is appropriate. Click OK.
5
Choose the default statistics: Mean 100% and M.S.D. 100%. The sliders
allow you to eliminate a certain percentage of outliers. 100% means
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that all spots are included, even outliers. Leave the default value and
click OK.
In the displayed window, you will see four columns. The first one displays the IDs
of the matches (i.e. the ID of the spot in the master gel). The columns AT1bis and
AT2bis contain the mean values (in terms of %Vol) of all the spots in the
corresponding class (for the particular match). The Max column displays the
highest value of the two means.
You can change the content of the columns when you select a different measure
in the Displayed value list at the top right corner of the Inter-Class Report window.
When you choose Dispersion for example, the values displayed in the columns
AT1bis and AT2bis correspond to the dispersion (M.S.D.) in each match. Again the
Max column displays the higher of the two values.
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52
6
Choose Ratio from the Displayed value list at the right of the Inter-Class
Report toolbar.
7
Click on the Save icon in the Inter-Class Report toolbar to save your
report.
8
Browse the folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial4 and
save the report with the name Inter-class _Report_Ratio.rpt (in Report
format). Click Save. The report is automatically included in the Reports
folder of the project.
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9
Choose the option Refine Selection in the Select on Gels drop down
menu.
10 Choose the Max column, and then click OK. Note that you can choose
one of the other columns (AT1bis or AT2bis). However, it is a good habit
to select the Max column so that the operation is generic (in particular
for scripting purposes).
11 Select rows having a Ratio higher than 2, then click OK.
12 Make a new Report From Selection. This feature can be accessed from
the Reports drop down menu.
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13 In the newly created Inter-Class Report, choose Gap in the Displayed
value list.
14 In the main program window, choose Window and select the previously
saved report (Inter-class_Report_Ratio.rpt) from the list at the bottom
of the menu. It will pop up in front of the other report. Close this report
(displaying the Ratios).
15 In the remaining Gap report, sort the values in descending order by
clicking once in the Max column header.
16 Click on the Annotate icon in the report toolbar to add an annotation
category in the report.
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17 Enter the name of a new category called Set:Verified. Click OK, and
again OK to accept the default category constraints and properties.
18 You will now have an extra column called Set:Verified in your report.
19 Select all rows in the gap report by clicking on the upper-left cell in the
table.
20 Pick the Inter-Class+Intra-Class Histograms option from the Histograms
drop down menu in the report toolbar. This displays a window with
histograms showing the individual spot values (orange bars) in each
class (separated by a gray vertical line). The horizontal blue line indicates
the mean value for each class, and the red lines define the dispersion
intervals.
21 Select the first line in the gap report (Inter-Class Report) and press the
Select on Gels icon in the report toolbar to see the corresponding spots
on your gels. If a spot belonging to the match is present on the gel, it is
displayed in the center of the cell in which the gel is displayed. If it is
absent in a gel, you can activate the Hand tool and double click on the
corresponding position in one of the other gels. All gel images will be
centered on this position.
22 Verify if the corresponding spots are properly matched. If this is the case,
and if the spot is differently expressed in the two classes, tick the check
box in the Set:Verified column of the Inter-Class Report window. To
indicate that the report has been modified, the corresponding cell
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becomes dark green (or gray, if the line was not selected) and an asterisk
appears after the window name.
23 Press the Select Next icon in the report toolbar to select the following
match in the report and on the gels.
24 If the match is of interest, that is, if the spot is differently expressed,
check the corresponding box in the Set:Verified column.
Sometimes, a more quantitative view of the spots is necessary to decide whether
a protein is differently expressed or not. The Inter-Class+Intra-Class Histograms
are helpful in that case:
25 Make sure the match you are interested in is selected in the gap report.
26 Use the Select on Gels+Reports option. This feature is available from the
Select on Gels drop down menu in the Inter-Class Report toolbar. Please
note that the histogram window needs to be docked to remain visible
(see Tutorial 1 to learn more about docking report windows).
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27 The corresponding spots are shown on the gels, and the histogram for
the selected match is highlighted in the Inter-Class+Intra-Class
Histograms window. If the values in the histogram clearly show that the
protein is differently expressed in the two classes, you can check the
corresponding box in the Set:Verified column of the Inter-Class Report.
You can also display a 3D view for the corresponding spots on your gels:
28 Select a line in your gap report.
29 Click on the Select on Gels icon.
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30 Choose Window > Mouse Selection > 3D View from the main program
menu.
31 A multiple 3D view is displayed (see details in Tutorial 2). Since the views
for the different gels use the same orientation and scale, you can easily
see the protein expression differences between the gels.
32 If you now click on the Select Next icon in the toolbar of the Inter-Class
Report, the spots of the next match are selected on the gels and directly
shown in the 3D View window (because you opened a mouse selection
3D View, see details in the manual).
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Systematically verify all the matches in the gap report, or stop when you
consider having enough protein spots to start working with. When finished, label
the interesting spots on the gels:
33 Select all rows in the gap report by clicking on the upper-left cell in the
table.
34 Click the Update Gels icon in the toolbar of the gap report.
35 Answer Yes to add the labels in one gel (the master gel).
On the master gel, you will now see spots with the label Verified. To easily mark
the corresponding spots on the other gels as well, so that they can be selected for
spot picking, for instance:
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36 Select all the gels in the active worksheet (Ctrl+A).
37 Choose Edit > Annotations > Copy Matched Labels in the main program
menu.
38 You will be asked if you want to propagate a certain number of labels
from 1 category (Set:Verified) to 6 gels. Answer Yes.
39 The software will tell you how many labels were added (some spots are
absent on certain gels, so no labels are created in this case). Click OK to
close the window.
40 All six gels will now have labels with the content Verified.
Save the gap report:
41 Click on the Save icon in the toolbar of the gap report.
42 Browse the folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial4 and
save the report with the name Inter-class_Report_Gap.rpt (in Report
format). Click Save. The report is automatically included in the Reports
folder of the project.
In order to confirm your results, you can repeat this analysis with the classes AT1
and AT2. These contain the same gels but use matches from a different match set
(Bacteria1).
In a next stage, you may also want to compare classes Abis and Bbis. To do so,
simply select these two classes and right click on one of them. Choose Open from
the contextual menu. The twelve gels are opened in two panes, as part of the
classes Abis and Bbis (and not as part of the four subclasses). Additionally, you
can study the protein expression differences between the treatment groups T1
and T2, independently of the substrates (substrate A and substrate B) used to
grow the cells. In this case, you can work with the classes T1 and T2 (Bacteria1b).
More generally, you can design any class that helps you to carry out your
analysis.
4.5
Pick list
To use the Ettan Spot Picker or Ettan Spot Handling Workstation, two adhesive
markers should be placed on the gel before scanning. These markers are used for
the calibration of the coordinates, that is, for determining the correspondence
between the X and Y positions of the analyzed gel image and the coordinates of
the actual gel located on the spot picker.
Once the gel has been digitized and analyzed with ImageMaster 2D Platinum, the
software can generate a pick list. This list contains the location, in pixels, of the
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center of each spot you wish to pick, as well as the pixel coordinates of the
centers of the two reference markers.
To export a file with spot coordinates for use by the spot picker, you first have to
open the image files and perform image analysis (spot detection is mandatory).
You should then annotate the reference markers. To do this exercise, two
detected gels with reference markers can be found in the Spot Picking project in
the Tutorial4 workspace:
1
Choose Select > Gels > All (Ctrl+A) in the menu.
2
Select File > Close All in the menu.
3
Select the gels SpotPick1.mel and SpotPick2.mel in the Gels folder of the
Spot Picking project by clicking on their names while holding the Ctrl
key.
4
Right click on one of the gel names and choose Open > In Worksheet in
the contextual menu.
5
Hide the workspace.
6
Identify the two reference markers.
If the reference markers are well detected (nice round spots perfectly centered on
the marker) during the automatic spot detection process, you can just add an
annotation on each of the marker spots. This is the case for both reference
markers in SpotPick1.mel and for the right marker in SpotPick2.mel:
7
Zoom the image SpotPick1.mel to better see the left reference marker.
8
Pick the Annotation tool in the program toolbar.
9
Double click on the marker spot.
10 Select the Comment category and click OK.
11 Enter IR1 as the label text and click OK.
12 The annotation will be linked to the marker spot and its coordinates will
be those of the center of the spot.
13 Repeat the procedure for the second (right) reference marker, but enter
the label text IR2.
14 Repeat the procedure for the right reference marker in SpotPick2.mel.
Its label must be IR2.
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If the reference markers are not well detected (irregular shape or split), as is the
case for the left reference marker in SpotPick2.mel:
15 Select the spot that is on the marker, using the Spot tool
16 Choose Edit > Spots > Delete. Answer Yes when you are asked for
confirmation.
17 Zoom the image to better see the left reference marker.
18 Pick the Annotation tool in the program toolbar.
19 Double click in the center of the reference marker.
20 Select the Comment category and click OK.
21 Enter IR1 as the label text and click OK.
22 The annotation is on the pixel where you double clicked. If this is not the
center of the reference marker, you must move the annotation by
clicking on its basis (cross) and dragging it to the middle of the marker.
You can now export a pick list with the included reference markers for each gel.
23 Select spots to pick. For example, select the spots belonging to the set
Pick by choosing Select > Annotations > By Category from the program
menu and selecting Set:Pick. Click OK .
24 Choose File > Export > Spots to Picker > GE Healthcare Ettan.
25 For each gel, you will be asked to save a pick list in text or XML format
(only the text file can be read by the Ettan Spot Picker). Enter a file name
and destination folder and click Save.
The Ettan Spot Picker or Ettan Spot Handling Workstation can read the exported
files. You can include the pick list files in your workspace, for later reference:
26 Right click on the Documents folder under the Spot Picking project in the
Tutorial4 Workspace.
27 Choose Add Files in the contextual menu.
28 Browse the appropriate folder, select the pick lists and click OK.
29 The pick lists now appear in the Documents folder.
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5
5.1
DIGE analysis
Introduction
If you purchased ImageMaster 2D Platinum DIGE, you will have access to all the
functionalities needed to efficiently and accurately analyze your DIGE gel
images. ImageMaster 2D Platinum DIGE in particular features the co-detection
algorithm developed by the GE Healthcare DeCyder software team.
NOTE! Please note that if the DIGE functions are absent or grayed out in the
software, your license is not valid for the DIGE module and you will not be able
to carry out the following tutorial.
In this tutorial, you will learn more about the specificities of a DIGE gel analysis.
Note that the general procedure, and most of the functionalities are the same as
those used for conventional 2-DE gels. Therefore, it is recommended to work
through the previous tutorials to get familiar with the most commonly used
features.
The present tutorial illustrates the analysis of a DIGE experimental design
incorporating an internal standard with several replicate samples. This tutorial
describes how to find proteins that exhibit statistically significant changes
between control and treated groups of bacterial cultures.
Four replicate gels are loaded with bacterial lysates as indicated in the table
below.
Gel number
Cy2
Cy3
Cy5
Gel 1
Standard
Control 1
Treated 1
Gel 2
Standard
Treated 2
Control 2
Gel 3
Standard
Control 3
Treated 3
Gel 4
Standard
Treated 4
Control 4
Each gel contains a standard sample to normalize control and treated samples
against. The standard sample is derived from the control and treated lysates,
which are pooled in equal concentration.
Tutorial gels
When you select Help > Tutorials > Tutorial 5 > Restore in the menu, any open
worksheets and workspace are closed and the Workspace window containing an
empty workspace is opened. In this tutorial, you can import the 12 GEL files (4
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DIGE gels, 3 images each) that can be found in C:\Program Files\GE
Healthcare\ImageMaster 2D Platinum\Tutorials\Tutorial5 (see directives below).
5.2
Create a new workspace and project
To create a new workspace:
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1
Click the Workspace tab below the ImageMaster toolbar to display the
Workspace window.
2
Click on the New icon in the toolbar of the Workspace window.
3
If you work on the tutorial gels, enter Tutorial5 in the Workspace Name
field, and browse the folder C:\...\ImageMaster 2D
Platinum\Tutorials\Tutorial1 to save your file. You can enter a comment
if you like. Click OK when you are finished.
4
Right-click on the workspace name (Tutorial5) at the top of the
workspace navigator and choose New Project in the contextual menu.
5
Enter DIGE as a new Project Name, a destination folder (Location) for
the project file (.prj) and possibly a Comment that describes the project.
Click OK.
6
You will find that your workspace now includes a project called DIGE,
containing the folders Gels, MatchSets, Classes, Reports and Documents.
If you purchased a license for ImageMaster 2D Platinum DIGE, a
subfolder called DIGE Gels exists in the Gels folder.
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5.3
Import DIGE gels
The images of each DIGE gel must be imported in the DIGE Gels folder in order to
create a DIGE gel entity.
1
Right click on the DIGE Gels folder in the workspace navigator. Choose
Import DIGE Gel in the contextual menu.
2
In the Import Image window, select the input format of the files and
indicate the reduction factor. For the provided tutorial gels, the reduction
factor should be 1 and the file format GEL. Then, click OK.
3
Browse the folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial5 and
select the image files Gel 01 Cy2 Standard.gel, Gel 01 Cy3 Control.gel
and Gel 01 Cy5 Treated.gel, while holding the Ctrl or Shift key. Click
Open to import the gel images.
4
The imported images are saved with the extension .mel. You can change
the file names manually, automatically add an extension to all the
existing file names, or decide to save the files in a different folder. For the
purpose of this tutorial, just click OK to save the .mel files in the proposed
folder.
5
Then, enter the name of the DIGE gel (Gel 01) and the dye chemistry
(minimal or saturation) that has been used (DIGE Min.) . You can also add
a comment concerning this DIGE gel. Click OK.
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6
In the next DIGE Gel Properties windows, enter the dye used for each
image, by choosing the right one in the proposed list. If the DIGE dye is
mentioned in the file name, the program gives it by default. Click OK.
7
Select the reference gel image among the different images of the DIGE
gel. Obviously, when an internal standard is used (often with Cy2), it
should be defined as the reference. Click OK.
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8
The DIGE gel Gel 01 appears in the DIGE Gels folder in the workspace
project. When expanding the gel by clicking on the
sign, the three
DIGE images become visible.
9
Repeat the procedure (steps 1 to 7) for the Gel 02, Gel 03 and Gel 04
image files.
5.4
Co-detecting DIGE gels
The co-detection algorithm is designed to simultaneously process the different
images derived from a single DIGE gel.
To perform spot detection on the four DIGE gels:
1
In the DIGE Gels folder of the project, select the four DIGE gels (Gel 01,
Gel 02, Gel 03 and Gel 04) by clicking on their names while holding the
Ctrl key.
2
Right-click on one of them, and choose Detect in the contextual menu.
3
The gel images are loaded and opened in a new worksheet.
4
In the DIGE Spot Detection window, enter an estimation of the Number
of Spots present on each image. For the current tutorial, choose 1500,
tick the Apply To All option and click OK.
5
A status window appears showing the progress of the spot detection.
Depending on your computer resources, the co-detection process can
take some minutes.
6
The spots appear on the gel images. Save them by choosing File > Save
> Worksheet in the menu.
7
Adjust the contrast of the open images using Show > Gels > Adjust
Contrast in the menu (see Tutorial 2).
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5.5
Displaying a DIGE histogram
When two co-detected images are selected, a DIGE histogram can be displayed.
It shows data associated with detected spots in the selected images. Spot data is
plotted against log volume ratio on the X-axis, using two Y-axes.
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•
The left Y-axis displays the spot frequency. The blue curve represents the
frequency distribution of the log volume ratios.
•
The right Y-axis represents the Measure parameter (Area, Max Volume, Max
Intensity or Max Slope) selected in the dropdown menu in the toolbar of the
DIGE histogram window. A plotted single data point on the histogram
represents an individual protein spot.
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To display a DIGE histogram:
1
Select two co-detected images (Gel01 Cy3 Control and Gel01 Cy5
Treated) by clicking on their legend, while pressing the Ctrl key.
2
Select the spots to be included in the DIGE Histogram. Generally, you
would select all spots in the images. So choose Select > Spots > All in the
menu (or use the Shift+A shortcut).
3
Choose Analyze > DIGE > Histogram in the menu.
4
A DIGE histogram containing all the selected spots is displayed.
5
In the drop down menu of the Settings icon, select Max Volume.
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6
Select several data points in the DIGE Histogram, by using the Ctrl key or
holding the left mouse button while drawing a (blue) region.
7
Click on the Select on Gels icon in the DIGE Histogram toolbar.
8
The corresponding spots will be selected on the images.
5.6
Displaying a DIGE report
A DIGE report is a special case of a spot report enabling you to display volume
ratios for specific combinations of DIGE images. Two co-detected images must be
selected to generate a DIGE report.
To display a DIGE report for the spots selected in the previous section (from the
DIGE Histogram):
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1
Select the DIGE Report option from the Reports drop down menu in the
toolbar of the DIGE Histogram window. Alternatively, if the two images
Gel01 Cy3 Control and Gel01 Cy5 Treated and some spots are still
selected, you can choose Analyze > DIGE > Report in the menu.
2
A DIGE report containing only the selected spots is displayed. You can
use the Settings icon to display only the columns of interest (see Tutorial
3).
3
The reported volume ratios (Vol Ratio) in the DIGE report are normalized,
so that the modal peak of volume ratios is zero (since the majority of
proteins are not up or down regulated). This means that the Vol Ratio is
expressed in the range of 1 to 1 000 000 for increases in spot volumes
and –1 to – 1 000 000 for decreases in spot volumes. Values between –1
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and 1 are not represented, hence a two-fold increase and decrease is
represented by 2 and –2, respectively (and not 2 and 0.5 as might have
been guessed). Please note that the DIGE report is the only occurence
where the Vol Ratio is expressed this way.
5.7
Creating DIGE match sets
In the following section, you will learn how to match the four DIGE gels of the
project. First, a match set containing the four DIGE gels must be created.
1
In the DIGE Gels folder of the Tutorial 5 project, select the four DIGE gels
(not their individual images) by clicking on their names while holding the
Ctrl or Shift key.
2
Right-click on one of the selected gels, and choose Create MatchSet in
the contextual menu.
3
In the Add DIGE in MatchSet window, enter DIGE as a name for the
created match set, and click OK.
4
Each of the following Create MatchSet windows corresponds to one of
the four DIGE gels, which will become sub match sets that contain their
three respective images. Keep the proposed names for all these match
sets (click four times OK).
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You will find that a match set DIGE has been created in the MatchSets
folder of the project, and contains the four sub match sets Gel 01, Gel
02, Gel 03 and Gel 04. Expand the sub match sets by clicking on the
signs. The red tick in the corner of the Control and Treated images
indicates that these images have been matched automatically with
their corresponding DIGE reference image (having a red component).
6
Right-click on the match set DIGE and choose Open from the contextual
menu.
7
You are asked to define a Reference among the four DIGE gels to create
the master for the match set DIGE. Choose one of the gels with the most
and best resolved spots. For this tutorial, choose Gel 03 and click OK.
8
In the Workspace window, the red component on the Gel 03 match set
indicates that it is used as a reference to create the Master of match set
DIGE.
9
Four master images representing the four sub match sets are opened in
a new [MatchSet] worksheet. These master images (Master_Gel 01,
Master_Gel 02, Master_Gel 03 and Master Gel_04) are created based
on the reference image (Standard) of each match set. Note that instead
of the Master_Gel 03, which is the Master of the reference sub match set
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Gel 03, the master Master_DIGE is shown. As the master and reference
images are the same, and matches are automatically created between
the two, the reference image is not displayed by default. It is possible to
display it by choosing Show > Show Reference in the menu. To prevent
confusion during matching, we recommend not to display the
Reference.
5.8
Matching DIGE gels
First position a landmark:
1
Make sure all spots are deselected by first selecting all gel images
(Ctrl+A) and then choosing Select > Unselect All in the menu (if the option
is grayed out, all your spots were already deselected).
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2
Using the SHIFT key, select the same spot on all gel images with the Spot
tool (choose small, well-defined, unambiguously corresponding spots
that are not in distorted regions).
3
Choose Edit > Annotations > Add Label in the menu.
4
Choose the category Landmark. Click OK.
5
Supply a name such as L1. Click OK to close the window, and Yes to
confirm that you want to add labels to your gel images.
To perform automatic matching:
6
Select all the images (Ctrl+A).
7
Choose Edit > Matches > Match Gels in the menu.
8
All the gels are matched to Master_DIGE.
9
When matching is completed, ImageMaster gives the total number of
matches found. Click OK to close this message.
10 Choose File > Save > Worksheet to save all modifications (annotations
and pairs) done in this worksheet.
To view the matching results:
11 Show the match vectors between the spots in the displayed images and
those in the Master by choosing Show > Matches > Show Vectors in the
menu.
12 Vectors are shown for all images but the master image. These vectors
are automatically minimized in the displayed region.
13 If the matching results are not satisfying, you can add additional
landmarks or manual matches. In order to optimize the matching, avoid
placing landmarks in distorded regions. Then re-run the matching
process (if you manually added matches, keep the existing matches
when asked for it).
14 You can also check the matches for individual spots using Select >
Matches > For Spots in the menu. This option selects all the matches
corresponding to the spots currently selected by the user.
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5.9
Creating classes with DIGE images
To find significant protein expression variations between the control and treated
samples, classes must be created for each type of sample.
To create a class for the Control samples:
1
In the Workspace window, expand the MatchSets Gel 01, Gel 02, Gel 03,
and Gel 04, and select all the images corresponding to Control samples
by clicking on their names while holding the Ctrl key.
2
Right-click on one of the selected gels, and choose Add In Class in the
contextual menu.
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3
In the Add Gels In Class window, enter Control as a name for the created
class, and click OK.
4
A class, called Control, is created in the Classes folder.
5
Repeat the procedure (steps 1 to 4) for the Treated samples.
6
Select the classes Control and Treated by clicking on their names while
using the Ctrl key.
7
Right-click on one of them, and choose Open in the contextual menu.
8
A pane is displayed for each class in the new [Class] worksheet. It is
possible to display the Master by choosing Show > Show Master in the
menu (or using the F1 key).
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5.10
Comparing classes with DIGE images
The comparison of classes containing DIGE images is very similar to the
comparison of classes with non-DIGE gels. The only significant difference is that
the Volume Ratio is generally the most appropriate value to use in your InterClass Reports because it uses the internal standard.
1
Choose Select > Gels > All (Ctrl+A) in the menu to select all the gels from
the classes Control and Treated. Only selected gels will be included in
the analysis.
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2
Select all matches by choosing Select > Matches > All (Ctrl + Shift +A) in
the menu. The spots matched to spots in the Master become green.
3
Choose Select > Matches > Refine Selection in the menu. Choose the
selected spots is >= option, and enter 6 with the scrolling bar. This
allows you to select only the matches that exist on 6 images or more.
4
Choose Analyze > Inter-Class > Report in the menu.
5
Select the value type to be used. In DIGE applications using an internal
standard, including this tutorial, the Volume Ratio is most appropriate.
Click OK.
6
Choose the default statistics: Mean 100% and M.S.D. 100%. The sliders
allow you to eliminate a certain percentage of outliers. 100% means
that all spots are included, even outliers. Leave the default value and
click OK.
7
Choose Ratio from the Displayed value list at the extreme right of the
Inter-Class Report toolbar.
8
Click on the Save icon in the Inter-Class Report toolbar to save your
report.
9
Browse the folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial5 and
save the report with the name DIGE_Inter-class _Report_Ratio.rpt (in
Report format). Click Save. The report is automatically included in the
Reports folder of the project.
10 Choose the option Refine Selection from the Select on Gels drop down
menu in the report toolbar.
11 Choose the Max column, and then click OK. Note that you can choose
one of the other columns (Control or Treated). However, it is a good habit
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to select the Max column so that the operation is generic (in particular
for scripting purposes).
12 Select rows having a Ratio higher than 2, then click OK.
13 Make a new Report From Selection. This feature can be accessed from
the Reports drop down menu.
14 In the newly created Inter-Class Report, choose Gap in the Displayed
value list.
15 In the main program window, choose Window and select the previously
saved report (DIGE_Inter-class_Report_Ratio.rpt) from the list at the
bottom of the menu. It will pop up in one of the docking areas, in front of
the other report. Close this report (displaying the Ratios).
16 In the remaining Gap report, sort the values in descending order by
clicking once in the Max column header.
17 Click on the Annotate icon in the report toolbar to add an annotation
category in the report.
18 Enter the name of a new category called Set:Verified. Click OK, and
again OK to accept the default category constraints and properties.
19 You will now have an extra column called Set:Verified in your report.
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20 Select all rows in the gap report by clicking on the upper-left cell in the
table. Dock this report window to avoid that it closes automatically.
21 Pick the Inter-Class+Intra-Class Histograms option from the Histograms
drop down menu in the report toolbar. This displays a window with
histograms showing the individual spot values (orange bars) in each
class (separated by a gray vertical line). The horizontal blue line indicates
the mean value for each class, and the red lines define the dispersion
intervals. Dock this histogram window to avoid that it closes
automatically.
22 Select the first line in the gap report (Inter-Class Report) and press the
Select on Gels icon in the report toolbar to see the corresponding spots
on your gels. If a spot belonging to the match is present on the gel, it is
displayed in the center of the cell in which the gel is displayed. If it is
absent in a gel, you can activate the Hand tool and double click on the
corresponding position in one of the other gels. All gel images will be
centered on this position.
23 Verify if the corresponding spots are properly matched. If this is the case,
and if the spot is differently expressed in the two classes, tick the check
box in the Set:Verified column of the Inter-Class Report window. To
indicate that the report has been modified, the corresponding cell
becomes dark green (or gray, if the line was not selected) and an asterisk
appears after the window name.
24 Press the Select Next icon in the report toolbar to select the following
match in the report and on the gels.
25 If the match is of interest, that is, if the spot is differently expressed,
check the corresponding box in the Set:Verified column.
Sometimes, a more quantitative view of the spots is necessary to decide whether
a protein is differently expressed or not. The Inter-Class+Intra-Class Histograms
are helpful in that case:
26 Make sure the match you are interested in is selected in the gap report.
27 Use the Select on Gels+Reports option. This feature is available from the
Select on Gels drop down menu in the Inter-Class Report toolbar.
28 The corresponding spots are shown on the gels, and the histogram for
the selected match is highlighted in the Inter-Class+Intra-Class
Histograms window. You may have to click on the docked tab of this
window to bring it to the front. You can also pin the window so that it
remains visible (see Tutorial1 for more details about dockable windows).
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29 If the values in the histogram clearly show that the protein is differently
expressed in the two classes, you can check the corresponding box in
the Set:Verified column of the Inter-Class Report.
You can also display a 3D view for the corresponding spots on your gels:
30 Select a line in your gap report.
31 Click on the Select on Gels icon.
32 Choose Window > Mouse Selection > 3D View from the main program
menu.
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33 A multiple 3D view is displayed (see details in Tutorial 2). Since the views
for the different gels use the same orientation and scale, you can easily
see the protein expression differences between the gels. Dock the 3D
View window.
34 If you now click on the Select Next icon in the toolbar of the Inter-Class
Report, the spots of the next match are selected on the gels and directly
shown in the 3D View window (because you opened a mouse selection
3D View, see details in the manual).
Systematically verify all the matches in the gap report, or stop when you
consider having enough protein spots to start working with. When finished, label
the interesting spots on the gels:
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35 Select all rows in the gap report by clicking on the upper-left cell in the
table.
36 Click the Update Gels icon in the toolbar of the Gap report.
37 Answer Yes to add the labels to one gel (the master gel).
38 Click on the Save icon in the Inter-Class Report toolbar to save your
report.
39 Browse the folder C:\...\ImageMaster 2D Platinum\Tutorials\Tutorial5 and
save the report with the name DIGE_Inter-class_Report_Gap.rpt. Click
Save. The report is automatically included in the Reports folder of the
project.
40 Save all the changes by choosing File > Save > Save All in the menu.
41 Close all the open reports by choosing Window > Close All in the menu.
42 Close all the open worksheets by choosing File > Close All in the menu.
5.11
Match with a preparative gel
When a fluorescently post-stained preparative gel is used for picking (including
reference markers), it can be imported as a DIGE gel even if it is a single image.
This allows to use the same DIGE spot detection algorithm as for the
corresponding DIGE gels. The detected preparative gel can then be matched to
the DIGE master to allow selection of spots to be picked.
1
Right-click on the DIGE Gels folder in the workspace navigator. Choose
Import DIGE Gel in the contextual menu.
2
In the Import Image window, select the input format of the files and
indicate the reduction factor. In this case, the reduction factor should be
1 and the file format GEL. Then, click OK.
3
Browse the folder C:\…\ImageMaster 2D Platinum\Tutorials\Tutorial5 and
select the image file Pick.gel. Click Open to import the gel image.
4
Click OK to save the .mel file in the proposed folder.
5
Then, enter the name of the DIGE gel (Pick). You can also add a comment
concerning this pick gel. Click OK.
6
In the next DIGE Gel Properties windows, choose Sypro Ruby in the
proposed list. Click OK.
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7
Right-click on the Pick DIGE gel and choose Detect in the contextual
menu. The gel image is loaded and opened in a new worksheet.
8
In the DIGE Spot Detection window, enter an estimation of the Number
of Spots present on the image. This detection parameter generally
should be close to the one used for the other DIGE gels. Choose 1500 and
click OK.
9
Save the changes made on the pick gel by choosing File > Save >
Worksheet in the menu.
10 In the workspace navigator, drag and drop the image Pick.mel within
the match set DIGE.
11 Right-click on Pick.mel within the match set DIGE and choose Open in
the contextual menu. This opens Pick.mel and the master of the match
set DIGE in a [MatchSet] worksheet.
12 Select the two gels.
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ImageMaster 2D Platinum Tutorial Edition AA
5
DIGE analysis
13 Choose Edit > Matches > Match Gels in the menu.
14 The image Pick is matched to Master_DIGE.
15 When matching is completed, ImageMaster gives the total number of
matches found. Click OK.
16 If the matching results are not satisfying (see Tutorial 3 for details about
matching validation), you can add landmarks on the pickgel and the
master and re-run the matching process to improve it.
5.12
Pick list
1
A pick list can now be generated:
2
Choose Select > Annotations > By Category in the menu.
3
In the Select Labels By Category window, pick Set:Verified in the
category list. Click OK.
4
Choose Select > Matches > For Spots in the menu. All spots in Pick.mel
matched to Verified spots in the Master_DIGE are selected. Make sure
the matches are correct.
5
Unselect the master image by clicking on its legend while holding the
Ctrl key.
6
Choose Edit > Annotations > Add Label.
7
In the Add Label window, create a category Set:Pick. Click OK, and again
OK to accept the default category constraints and properties.
If the reference markers are not well detected, as is the case for the present pick
gel, you can delete the spots and just define an annotation to mark the position
of the reference markers:
8
Select the spots that are on the markers.
9
Choose Edit > Spots > Delete. Answer Yes when you are asked for
confirmation.
10 Zoom the image to better see the left reference marker.
11 Pick the Annotation tool in the program toolbar.
12 Double click in the center of the reference marker.
13 Select the Comment category and click OK.
ImageMaster 2D Platinum Tutorial Edition AA
87
5
DIGE analysis
14 Enter IR1 as the label text and click OK.
15 The annotation is on the pixel where you double clicked. If this is not the
center of the reference marker, you must move the annotation by
clicking on its basis (cross) and dragging it to the middle of the marker.
16 Repeat steps 10 to 15 for the right marker but enter IR2 instead of IR1 as
a label for the annotation.
You can now export a pick list including the reference markers.
17 Make sure that only the pickgel is selected.
18 Select the spots belonging to the set Pick by choosing Select >
Annotations > By Category from the program menu and selecting
Set:Pick. Click OK .
19 Choose File > Export > Spots to Picker > GE Healthcare Ettan.
20 You will be asked to save a pick list in text or XML format (only the text
file can be read by the Ettan Spot Picker). Enter a file name and
destination folder and click Save.
The Ettan Spot Picker or Ettan Spot Handling Workstation can read the exported
files. You can include the pick list files in your workspace, for later reference:
21 Right click on the Documents folder in the Tutorial5 Workspace.
22 Choose Add Files in the contextual menu.
23 Browse to the appropriate folder, select the pick lists and click Open.
24 The pick lists now appear in the Documents folder.
88
ImageMaster 2D Platinum Tutorial Edition AA
This version of ImageMaster has been developed by the Swiss Institute of
Bioinformatics in collaboration with GeneBio and GE Healthcare.
All intellectual property rights on this Tutorial, as well as on the ImageMaster 2D
Platinum software, belong to the Swiss Institute of Bioinformatics. No part of
this Tutorial may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopy, recording or any information
storage or retrieval system, without permission in writing from the Swiss
Institute of Bioinformatics.
© 1992-2005 Swiss Institute of Bioinformatics - All rights reserved.
Swiss Institute of Bioinformatics
CMU-Rue Michel-Servet 1, CH-1211 Geneva, Switzerland
ImageMaster 2D Platinum provides access to several databases on the
Internet. It is the responsibility of the user to acquire the database licenses, if
needed. In particular, the PROSITE and SWISS-2DPAGE databases are
copyright, and all commercial users of these databases are required to
purchase a database license from Geneva Bioinformatics (GeneBio) SA. Please
contact GeneBio at [email protected] for more information.
Geneva Bioinformatics (GeneBio) SA
Avenue de Champel 25, CH-1206 Geneva, Switzerland
ImageMaster 2D Platinum uses the DeCyder co-detection algorithm.
© 2005 General Electric Company – All rights reserved.
ImageMaster 2D Platinum uses the TIFF library.
© 1988-1999 Sam Leffler and 1991-1999 Silicon Graphics, Inc - All rights
reserved.
ImageMaster 2D Platinum uses software developed by the Apache Software
Foundation (http://www.apache.org).
© 1999-2003 The Apache Software Foundation - All rights reserved.
www.amershambiosciences.com
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Amersham Biosciences AB
Björkgatan 30
751 84 Uppsala
Sweden
Cy, CyDye, Ettan, Typhoon, DeCyder, ImageMaster, LabScan and
ImageScanner are trademarks of GE Healthcare Ltd. GE tagline and GE
monogram are trademarks of General Electric Company.
SYPRO is a trademark of Molecular Probes Inc.
Macrovision is a registered trademark and FLEXIm is a trademark of
Macrovision Corporation. ExPASy is registered by the Swiss Institute of
Bioinformatics. Microsoft, Windows, PowerPoint and Microsoft Internet
Explorer logo are trademarks of Microsoft Corporation. Netscape Navigator is a
trademark of Netscape Communications Corporation. TIFF and Photoshop are
trademarks of Adobe Systems Incorporated.
All goods and services are sold subject to the terms and conditions of sale of
the company within GE Healthcare which supplies them. GE Healthcare
reserves the right, subject to any regulatory and contractual approval, if
required, to make changes in specifications and features shown herein, or
discontinue the product described at any time without notice or obligation.
Contact your local GE Healthcare representative for the most current
information.
Amersham Biosciences AB, a General Electric company going to market as
GE Healthcare.
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