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DeCyder Image Analysis Tutorial (contact Jim Malone at (314) 286-2452 for access to DeCyder DIA)

DIA Analysis of features within a single gel

 

1) Loging In – Log into one of the data computers in room 721 Southwest Towers using “Visitor” as the username and “proteins” as the password. Double click on the gray DeCyder 2D Icon on the desktop. In the DeCyder login window enter the Username “Core” and Password “MALDI5” and left click on the “More” button. If the box at the lower left corner of the window has a checkmark, left click on the check to remove it then left click on the Login button.

 

2) Create a Workspace – Open the DIA software by left-clicking on the Differential In-Gel Analysis icon in the Decyder window.  Select “Create Workspace” from the "File" menu to set-up a new analysis. In the “Create Workspace” window scroll to your PI name and double-click on the icon to the left of the PI name. A “gel” icon should appear: double click on it to show the gel image sets present in DeCyder. Left click on the gel of interest and then left click on the “Create” button to create a new workspace. Although your data set may contain up to three images, only two will be displayed at a time.

 

3) Process Gel Images – Select “Process Gel Images” from the “Process” menu. In the “Process Gel Images” window which opens check to see that the Algorithm Selection shows “DeCyder detection algorithm 6.0”. If it does not say this, click on the arrow at the right and select algorithm 6.0. Change the number in the “Estimated no. of spots” blank to equal 10000 then click the “OK” button to detect spots.

 

4) Select an Area of Interest – When spot detection is complete, the gel images will show multiple red, green, and blue spot boundaries. Select “Properties” from the “View” menu and click on the “Table View” tab. Click boxes as necessary so that only the “Picked spots” and “Protein of Interest spots” boxes are checked, then click on the “Spot Display” tab. Click boxes as necessary so that only the “Picking references and pick locations”, “Picked spots” and “Protein of Interest spots” boxes are checked, then click the “OK” button.  Select “Define Area of Interest” from the “Edit” menu. Move the rectangular target pointer to the upper left corner of the area you wish to analyze. Left click and hold as you drag the arrow to the lower right corner of the area of interest. Release the left mouse button and the area of interest should be outlined on both of the images. Using this approach, all spots lying outside of the area of interest will be removed when the exclusion filter is applied. It is not critical that the three reference spots are within the area of interest.

5) Automated artifact exclusion – S elect “Exclude Filter” from the “Process” menu. In the “Exclude Filter” window click on the box to the left of “Volume” and enter “10000” in the space to the right. Click “OK” and the exclusion filter will exclude all spots outside of the area of interest, and those with volumes of less than 10000. Excluded spots are still present and may be restored for analysis, but while excluded, they will be ignored by the software. Select “Properties” from the “View” menu and click on the “Table View” tab. Click the boxes to the left of “Decreased” and “Increased” then click on the “Spot Display” tab. Click the boxes to the left of “Decreased” and “Increased” then click “OK”. View the “Histogram selections” section at the upper right corner of the screen. Set the “Threshold mode:” to manual, then enter a threshold value equal to the number to the right of the “2SD:” value shown just below the threshold. Hit the "Enter Key to make the threshold change.

6) Manual artifact exclusion - The images (and the table at the lower right part of the desktop) now only show increased and decreased features outside of 2SD of the normalized distribution. Spots which are greater in the right image have blue outlines while those which are smaller in the sample on the right are represented with red spot boundaries.

Image analysis software does not recognize all 2D gel artifacts. Manual inspection is required to select gel features for protein identification. Select a "decreased" or "increased" gel feature from the table, and examine the 3D view showing the feature from both gel images. The following guidelines are examples of common features seen on 2D gels.

Resolved protein spots appear as symetrical peaks with rounded edges and smooth sides. These features are usually single protein forms. Comparing the left 3D image to the right, it is obvious that there is a significant difference in peak volume.

 

 

A 2D gel may have streaks (horizontal or vertical) consisting of unresolved proteins or isoforms. Both quantitation and protein identification is compromised for these features, therefore they should not be selected for excision.

 

 

Relative quantitative differences are affected by saturation of the digital image during gel scanning or subsequent image analysis. These examples of a high abundance protein will not be quantified correctly because data is missing for each peak. These features may be selected for excision, but it is impossible to determine the significance of differences between these samples without re-scanning the gel images.

 

Particulates generate sharp spikes in gel images. These spikes typically have very straight sides from the base to the top, unlike the curving sides apparent on protein peaks. Elimination of these features from the images improves the quality of normalized data. These features should not be selected for excision.

 

 

 

Background noise typically appears as jagged surfaces on peaks and flat areas. Although the two peaks shown here appear to be approximately the same size, the peak on the left may seem significantly larger because of the background fluctuations. Although the background differences do not impact protein identification, they affect quantitation, and would require running an additional gel with different dye combinations to validate differences.

The DeCyder DIA software uses an auto-ranging function to determine the maximum value shown in the 3D image. In this example, an adjacent high abundance protein makes the smaller feature difficult to evaluate.

 

 

 

 

 

Reducing the spot margin setting for displayed spots (in the "3D View" tab of the "Properties" selection of the "View" pull-down menu) makes it possible to view peaks which are near larger features. In the previous image pair the spot margin setting was 80. Reducing this setting to 3 allows the view at the right.

 

To see the spots better, it may be useful to zoom in on the gel images at the upper left corner of the screen. Click on the "Zoom in" button (a magnifying glass with a plus sign next to it) to increase the zoom level. Repeat as necessary. To reduce the zoom, click on the "Zoom out" button (a magnifying glass with a minus sign next to it). To zoom fully out, click the "Fit to window" button (a magnifying glass with a rectangle next to it). To change the brightness and contrast of the images, select "Contrast/Brightness" from the "View" menu. Adjust contrast and brightness on each image as needed.

In cases where there are groups of spots which you wish to remove (like where there is a strong streak through the gel) select “Define Exclude Area” from the “Edit” menu and drag a box around those spots. All spots within this box will be excluded.

Click on each spot number in the table individually, and view the 3-D image to the left of the table. To rotate the 3-D image, move the mouse arrow inside of the 3-D image frame, then click and hold the left mouse button and drag the mouse to the left or right. Dragging the mouse up and down tilts the 3-D image down and up. Combining these movements makes it possible to view the 3-D images from any angle.

To remove artifactual features select the feature on the table and click the box marked “Exclude” at the bottom of the screen (placing a check in the box).

 

Change one image and repeat the process of manual exclusion for this image pair. Repeat this process one final time for the last image pair.

 

Once all artifactual features are marked for exclusion, select “Re-Normalize” from the  “Process” menu and re-adjust the threshold value as detailed in section 5 above.

 

 

7) Selecting Spots for Identification –Repeat the review process from the previous step for each spot increased or decreased by greater than 2SD. During this review, select spots to be picked by clicking the "Pick" box beneath the table. Review the “picked” spots in the images. The final list of picked spots should include no more than 24 spots. If more than 24 spots are chosen, Proteomics core personnel will remove picked spots to reduce the total of picked spots to 24. Alternatively, if you desire more than 24 spots cut from the gel, we can break your picklist into multiple lists of up to 24 spots each.

8) Submitting your analysis to the Proteomics Core Facility - Once you have completed the DeCyder analysis, save the workspace by selecting "Save Workspace as" from the "File" menu. Enter a name for this analysis by clicking in the space to the right of “Workspace name:” near the bottom of the “Save Workspace” window. Click the “Save” button to save the analysis. Contact us at jmalone@proteomics.wustl.edu to request that we cut and analyze the spots on your spotlist.  In this communication, please include the name of the workspace file and the PI for whom the work was done.