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

BVA analysis of multiple gel images

1) DIA Analysis: In order to perform analyses using the BVA software module, images from a each gel need to be analyzed first using the DeCyder DIA software.

2) Setting up a BVA analysis: Beginning at the DeCyder 2D menu, click on the Biological Variation Analysis logo beneath this title. Select “Create Workspace” from the File menu.

Scroll through the list of investigators to reach your PI’s name. Double click on the name to show DIA analyses available for BVA. Holding down the control key, click on each of the gels to be included in the BVA analysis, then click the Add button to the right. Click Create and BVA will enter all gel images into a new BVA file and select a Master image containing the greatest number of spots.

3) Getting oriented in BVA: BVA is set up around 4 different “Tables” representing 4 phases of the BVA anlysis. Two letter abbreviations for each table appear above the gel images as ST, MT, PT, and AT. The Spot Map Table (ST) is used to group images, select the master image, and assign pick images. The Matching Table (MT) may be used to set landmark matches, promote automated matching, and edit matches. The Protein Table (PT) displays quantitative data for matched gel features, detailing several quantitative measures for individual spots. The Appearance Table (AT) displays the quantitative data from a single gel feature, across the entire set of gel images.

4) Grouping images: In order for BVA to compare quantitative information from different research conditions, each image must be assigned to a group. Images from experiment-wide protein pools should already appear in the Standard folder. These images are used for matching, and for ratio metric comparisons. The remaining groups need to be defined and gel images assigned to each. To define a new group, click the add button near in the upper right box, activating the “add group” function.  Enter a group name, and a brief description if desired. Condition 1 and Condition 2 may be assigned if you will be performing 2-way ANOVA on the data. Entries here should be in the form of a single digit number for each condition. (For a more complete understanding of how to use this function, refer to the DeCyder 2D Software manual). To change the color for the group, click on the color dot. Select a color by clicking it , then select “OK” to save the choice. Once all information about the group is in place, click the “Confirm” button to generate the group. Repeat this process for additional groups. To move gel images into groups, click on the “Unassigned” folder icon at the top center of the desktop. The window to the right of this will display all gel images which have not been assigned to a group. Select images to be assigned to a group by holding down the control key and clicking on each image you wish to include, then drag the group of images to the desired folder on the left. Repeat this process until all images are assigned to a group. (****Note****groups will need to contain at least 3 images each in order for any statistics to be performed on the group).

5) Activating Match Table and Setting Landmarks: Matching has already been performed on all images within individual gels, so BVA only requires matching of the pooled images to compare everything. Click on the blue MT at the top of the window, to activate the match table view. Click on the pull-down menu at the top of the images on the left and select the one with STANDARD in the name. This will be the Master image to which all other STANDARD, images are compared. Click on the pull-down menu at the top of the right image and select a different gel image which has STANDARD in the name. Click on the “Landmark mode” button at the bottom of the window.

Locate a spot in the Master image which has a counterpart in the second image. Click on the spot in the Master image, then click on the same spot in the second image to select a landmark which is the same in both images.  When the second spot is clicked, a line (vector) should appear indicating how far and in what direction this spot is from where it appeared in the first image. Generate additional landmarks using the method detailed here, distributing them evenly across the images. About 3-5 landmarks should be enough for successful matching. In every case, click on a spot on the left image before you click on the right image. If you accidentally select the wrong spot on the right image, wait until the vector is displayed, then click on the correct spot in the right image and DeCyder will fix the mismatch. Change the second image to another STANDARD image and repeat the landmarking process. It is not necessary to use the same landmark pairs on all images. Repeat this process until all STANDARD images are landmarked. Click on the Landmark mode button once again to de-select it.

6) Matching and Match editing: Select “Match” from the Process pull-down menu. Select “Match all” then click on Match at the bottom to begin the automated matching process. BVA will now match all images to one another. When the matching process is complete, BVA will return you to the Spot Map Table view. Click on the blue “MT” at the top of the screen to return to the Match table. Review the quality of the matching by viewing the match vectors (the lines mentioned above to verify that they are generally parallel for adjacent spots. If vectors are not visible, select “Properties” from the “View” pull-down menu. Place a check in the square at the bottom of the “Spot Features” box where it says “Match vectors when match table is displayed”. If there are spots which were incorrectly matched, these may be unmatched by clicking on the matched spot in the master image, then clicking the ”Break Match” button at the bottom of the screen. To generate missed matches, click on the spot in both images, than click the “Add Match” button at the bottom of the window.

In some cases, a single spot in one image may be segmented to appear as multiple spots in the other image. In this case, click on the matched spot matched (or match the single spot to one on the multi-spot gel), then select Merge Spots from the pull-down Edit menu. Adjacent unmatched spots will appear. Click on a spot and it will be added to the original spot. Repeat this process to combine additional spots if necessary.

The graph view seen in the upper right quadrant of the DeCyder window may be set to show how abundance values change for a selected spot in different groups or conditions. This representation is only available in Protein Table and Appearance Table modes. To change what is displayed in the scattergrams, select “Properties” from the “View” pull-down menu, and make selections from the listings shown under the Graph View tab. Abundance values show amounts of protein relative to the least abundant sample (setting this value to 1.0) while Standardized abundance shows amounts relative to the master image (setting this value to 1.0). **Note**All statistical analyses are performed using standardized log abundance values.

After matching and subsequent editing is complete, statistical analysis may be performed based on the original groupings. DeCyder generates Average Ratio, Student’s T-test analysis, and ANOVA analysis (one- and two-way) for each spot showing how it is represented throughout the image series.