Analyzing Gels with DeCyder 2D 6.0

The first step in analyzing any gel is to use the DIA program. No matter what the goal of the analysis is, you must create a spot map in DIA. To do so, you must begin by importing the gel into the DeCyder interface.

 

-Importing Gels

• Before entering DeCyder, make copies of the gel files (UNSEP 1, UNSEP 2, UNSEP 3)
• Rename the files with the following conventions
• Begin with “Gel ##” – for each file common to the first gel, start with “Gel 01”, for each file common to the second gel, start with “Gel 02”, etc.
• Then type the dye used to scan (Cy2, Cy3, Cy 5)
• If the file has an internal standard, type “Standard”
• End the name with any extra useful information, such as gel number, in parentheses
• Ex: Gel 02 Cy2 Standard (12345).gel
• Ex: Gel 01 Cy3 (56789 Sypro).gel
• At the main DeCyder screen, click on Image Loader
• Click add, and select the files to import. Repeat for each file (Tip: If files are in one folder, you can use the Ctrl key to select them all and add them at the same time)
• Select the project to import the files to. If a project has not yet been created, do so by clicking the file cabinet icon next to the Import button. Name the project and UNCHECK the public access box.
• Click Import

 

-Creating a Spot Map in DIA

• At the main DeCyder screen, click on Differential In-gel Analysis
• Go to File Create Workspace, or click the Create Workspace icon (it is located under the file menu and looks like a blank sheet of paper)
• Click the plus sign next to your project name, then click the plus sign next to the GEL folder.
• Select the Gel you wish to analyze. The images for that gel should appear on the right side of the menu. Click Create to analyze those images (to select only some of the images, you must manually select them using the Ctrl key and then click Create)
• When the images load, select the Contrast/Brightness icon and adjust the sliders so that all the spots can be seen on the gel as clearly as possible. Optimally, the image should look something like that in Figure 1 below.

• Click on Process Process Gel Images. Leave the estimated number of spots at 2500, and do not autodetect picking references.
• Click on the Properties icon (to the left of the print icon). Click on the Spot Display tab, and deselect Excluded Spots. (I prefer to view the selected spot only, so I also deselect Similar, Increased, and Decreased, but this is only a matter of preference.) Click on the Table View tab, and deselect Excluded Spots and Unconfirmed Spots (this makes filtering much easier).
• Go to Edit Define Picking Reference. Zoom in on the LEFT picking reference and click on the center of it such that the symbol fits the reference as best as possible. Then do the same for the RIGHT picking reference.
• Go to Edit Define Area of Reference. By dragging a rectangle from top-left to bottom-right, select the area you want to analyze. This should look something like the rectangle pictured above in Figure 1.
• Go to Process Exclude Filter. Click OK. This will exclude the spots outside the Area of Interest.
• In the table, click on the Max Slope header to sort the spots in order of decreasing slope. Scroll down through the spots until you find a spot that is a true protein (not a dust spot or other artifact). As for every filter, be conservative – if a spot might be a true protein, include it as one for now. Take the value of the slope of the first true protein and add .01. Go to Process Exclude Filter, check the slope box, and enter this value into the corresponding box and click OK.
• Click on the Area header in the table to sort the spots in order of increasing area. Scroll down through the spots until you find a true protein spot. Enter this value of area in the Exclude Filter and click OK. Repeat for Max Peak Height (increasing order), and volume (increasing order). When entering the volume filter, use the volume of the spot one above the true protein spot, to compensate for rounding in the table.
• If there are multiple gels for this experiment, each with an internal standard, DIA analysis is done for this gel. Repeat for the other gels in the experiment, and then analyze the gels with BVA.
• If there is only a single gel, or if there are no internal standards for this experiment, continue analyzing the gel in DIA.

 

-Using DIA to Analyze Single Gels or Gels with no Internal Standard

• Click on the Threshold Mode Pulldown Menu at the upper right of the screen. Set this to 2 Model S.D.
• Click on the Properties Icon, go to the Table View tab, and deselect Similar. Only the spots that Increased or Decreased should remain in the table.
• Look at each spot in the table. Click Confirm for every spot that is a true protein. These spots are proteins that are showing changes in expression at a 2-SD threshold.
• If more than 15-20 decreased or increased spots were found to not be true protein spots, exclude all of these. Take note of the current value for the threshold and go to Process Re-Normalize. The threshold may have been too loose; this will make the bound stronger (though not necessarily tighter).
• Sort the spots in order of average ratio. Look at any spots with an average ratio smaller than that of the original threshold (these will be new) to see if they are true proteins. If they are, confirm them. Do this for both the increased and decreased spots.
• If there is only one gel in the experiment, these spots will constitute your final list of spots with a change in expression. To create a pick list in DIA, see Creating a Pick List in DIA.
• If there is more than one gel in the experiment, analyze each gel in this manner. You can then use BVA to match the gels and select those spots that only show a 2-SD change in expression in each gel. Follow the instructions for using BVA in this manner here.

 

- Creating a Pick List in DIA

• For each spot you want to pick, check the pick box.
• Go to File Export Pick List
• Save the file so the pick list can be identified by its name (e.g., Gel XXXXX Pick List)

 

- Creating an Analysis for Gels with Internal Standards in BVA

• At the main DeCyder screen, click on Biological Variation Analysis
• Go to File Create Workspace, or click the Create Workspace icon (it is located under the file menu and looks like a blank sheet of paper)
• Click the plus symbol next to the workspace that contains your DIA files
• Click DIA. The list of DIA files for each gel in that workspace should appear in the middle window.
• Select the gels you want to include in the analysis and select Add. Then click Create.
• The program will automatically select the gel with the most spots as the Master Gel, the gel that all other gels will be matched to. Unless that gel is very difficult to see, this is the proper choice.
• In the experimental design view, there will be an Unassigned Folder and, if the original files were correctly named, a Standard Folder. If the standard folder is there, it should contain the internal standards for each gel. Check to be sure this is the case.
• If there is no Standard Folder, you must add one. Click Add, and name the group “Standard”. You must also create folders for the Cy3 and Cy5 samples. Click Add and give these folders appropriate names (Cy3 and Cy5, Control and Treated, etc)
• Click the Unassigned folder. The gels should appear in the middle window. Select the images you want to put in each folder (use Ctrl for selecting multiple images) and drag them into the correct folder to the left.
• You must now match the gels.

 

-Matching Gels in BVA

• Click the MT button on the toolbar. This will bring you to the match table screen. Generally, it is easiest to match the gels by making the spot map take up the full screen, so click the button on the toolbar that shows the upper left quadrant of a square being emphasized (or go to View Image View).
• The fastest way to match the gels is to match them all at once. To do so, go to View Display Multiple Gel Views and select one of the options. You will want one window for each gel (e.g. for 5 gels, select 2 X 3).
• Use the pulldown button at the upper right of each window to select the image of the internal standard image for a gel (all spots must be as visible as possible). View each gel in one window; if you have extra windows, ignore them.
• Before the gels are matched, they must be landmarked. Select a spot in the master gel and find it in as many of the other gels as possible, selecting it in each of those. Then select Add Match. To correct a mismatch, select the spot in the master gel and select Break Match

Tips for Matching Gels Accurately

• It is often easier to match the spots if the screen is less cluttered. Rather than viewing the outline of the spot, it may be easier to just view the location of each spot. To do so, click the Properties button on the toolbar (to the left of the print button) and go to the Image View tab. Uncheck the box labeled Signature.
• Begin with a spot or small group of spots that is large and easily seen on every gel. Use these beginning spots as a benchmark for matching other spots nearby and slowly spread the matches across the gel.
• Use the 3D View to confirm matches as you select them. The views may not look exactly the same, but the surrounding spots should be in the same general location in each gel. The 3D view shows the master gel, which is highlighted across the top in red, and the primary match gel, which is highlighted across the top in blue. To change which gel is selected as the primary match gel, click on the corresponding spot in the desired gel.
• If a spot in one gel appears as two spots in another gel, view the spot in the 3D View. If there is only 1 apparent peak, select one of the spots and go to Edit Merge Spot. Click the spot you want to merge to the selected spot. Then match the spots. If there are 2 peaks visible, match the lone spot to what appears to be the main peak of the two.
• Look at the match vectors as the matching proceeds. They should point in essentially the same direction throughout the gel, or else show a trend of slight change from top to bottom or left to right. If a match vector does not fit the general pattern, it is likely the match is incorrect.
• Try to match as many smaller and less conspicuous spots as possible. This will make the computer matching more accurate.
• Spread your landmarks across as much of the gel as possible. This will also aid in the automatic matching process.

• When all the gels are landmarked, go to Process Match. Select Match All and click Match.
• Check each gel for inaccurate matching. First, look at the match vectors. They should be pointing in the same general direction. Look for vectors that do not follow the main pattern. Break any incorrect matches and add the correct matches. Incorrect matches at the edges of gels can be ignored, but be sure to confirm these matches later if it is found that a protein showing significant change is located near the edge of a gel.
• Look for any noticeable unmatched spots – those where a spot is clearly visible but is not matched. In some cases, this is merely a match that the program did not make for one reason or another, and it can be easily added. In other cases, this has occurred because of incorrect matching in the area. If this is the case, correct the matches in any affected gels.
• The next step is to run a statistical analysis on the matched gels.

 

-Performing Statistical Analysis in BVA

• Statistics can only be calculated on gels with internal standards.
• The experimental design must first be set up as described here.
• Go to Process Protein Statistics. Choose paired tests if the samples in each gel are from the same animal (each gel is a different animal but the samples in any particular gel are from the same animal, such as a before and after test), otherwise choose independent tests. Select Average Ratio and Student’s T-test, and select the two groups you wish to compare. Click Calculate.
• The statistics will be displayed in the Protein Table, which can be viewed by clicking the PT button in the toolbar.
• To find the spots that show statistically significant change, order the table in order of increasing T-test. View each spot with a t-test smaller than 0.05. If a spot is a true protein, mark it as a protein of interest (POI). If not, ignore it.
• Click the Properties button on the toolbar (to the left of the print button). Click the Protein Table tab, and select Protein of Interest in the Protein Table Filter. This will eliminate all spots that were not true proteins or had a t-test greater than 0.05.
• From the proteins remaining, choose which spots should be picked and create a pick list, following the directions below.

 

-Creating a Pick List in BVA

• Go to the spot map table (click ST on the toolbar) and select the gel you wish to pick from. Click the Pick check box at the bottom of the screen.
• Go to the protein table (click PT) and click the Pick checkbox for any spots you wish to pick. To pick every spot, go to Protein Assign All Protein of Interest as Pick. Make sure the spots you wish to pick are matched in the gel you wish to pick from.
• To make multiple pick lists, click Change List at the bottom of the protein table screen. Select another list (change the name if you like) and click OK.
• To pick from multiple gels, mark each gel as a pick gel in the spot map table. After making a pick list for the first gel, change the list as described in the step above, and then use the dropdown menu directly below to change the spot map. Follow the same procedure as before to make a pick list for the second gel.
• To export the pick list, go to File Export Pick List. Select the list you would like to export and the corresponding spot map. Click OK. Repeat for each pick list you have created.

 

-Showing Annotations in BVA

• To label spots in the spot maps, click the Properties button in the toolbar (to the left of the print button).
• Click on the Image View tab. Check the box labeled Annotation. To show the annotations only for spots which are POI or are being picked, check the Filter Annotation Box and select the appropriate category from the dropdown menu.
• To move the annotations so that they are more easily seen, go to Edit Move Annotations, and drag each annotation to its desired location.

 

-Using BVA to Analyze Gels with No Internal Standards

• No statistical analysis can be performed on gels if there are no internal standards.
• However, the gels can be matched, and spots that show a change of at least 2-SD on each gel can be identified.
• Create a record of which spots show a 2-SD change in each gel.
• At the main DeCyder screen, click on Biological Variation Analysis
• Go to File Create Workspace, or click the Create Workspace icon (it is located under the file menu and looks like a blank sheet of paper)
• Click the plus symbol next to the workspace that contains your DIA files
• Click DIA. The list of DIA files for each gel in that workspace should appear in the middle window.
• Select the gels you want to include in the analysis and select Add. Then click Create.
• The program will automatically select the gel with the most spots as the Master Gel, the gel that all other gels will be matched to. Unless that gel is very difficult to see, this is the proper choice.
• Open the DIA file for the master gel, and record the coordinates for each spot that shows a 2-SD change in expression for that gel.
• Match the gels in the usual manner (see instructions here).
• Go to the match table, and order the table by the coordinates of the Master Gel.
• Using the master coordinates, find each spot that showed a 2-SD change in the DIA for the master gel.
• For each spot, select it and look at the 3D View window. For the selected gel in the Match window, the 3D View will show the DIA number that matches this spot. Compare this spot number to the list of spots you recorded from the DIA file for this gel. Do so for each gel. If the DIA numbers match, then the spot showed a 2-SD change in multiple DIA gels. These spots are good candidates to be picked – to do so, follow the instructions here for creating a pick list in BVA.

 

-Adding a New DIA File to a BVA Workspace

• Go to File Add Template/DIA Workspace. Perform the same steps as were needed to create the BVA workspace (see here).
• The new spot map may have more spots than any of the other spot maps. If the gels have not yet been matched, select this spot map in the spot map table and check the box labeled Master at the bottom of the screen to make this the master gel. If matching has already been done, it is best to leave the current master gel as the master. Otherwise, all of the matching will need to be redone.
• Match the new gel to the master gel and perform statistical analysis as described earlier.