1. Methodology for the analysis of the data of the separated proteome
“The proteome obtained by the 2-DE process must be analyzed to identify the significant spots by comparing the control and the patient or treated sample. It offers a flexible solution for the comprehensive visualization, exploration and analysis of 2D gel data”.
Related LOs: Scanning property, Statistic data analysis
> Prior Viewing – IDD-17. SDS-PAGE, IDD-20. Silver staining
> Future Viewing – IDD-26. Spot picking, IDD-27. In gel digestion, IDD-31. MALDI-TOF data analysis.
Course Name: Methodology for the analysis of the data of the separated proteome
Level(UG/PG): PG
Author(s): Dinesh Raghu, Vinayak Pachapur
Mentor: Dr.Sanjeeva Srivastava
Title of the concept
*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license 1

2. Learning objectives 1
After interacting with this learning object, the learner will be able to:
Identify/ Analyse Protein spot detection along with manual editing.
Practice matching and alignment of gels.
Analysis of data with statistical significant.
Infer the steps involved to perform the experiment.
Assess the troubleshooting steps involved in the experiments. 2 3 4 5 2

3. Image processing (Slide: 8-10)
Master Layout 1
Gel Scanning (Slide:5-7)
Image processing (Slide: 8-10) 2
Spot Processing (Slide: 11:14)
Gel matching (Slide: 15-17) 3
Statistical data analysis (Slide: 18-21) 4 5 3

4. 1 Definitions and Keywords 2 3 4 5
1. Smooth: Set this parameter first. It fixes the number of times ImageMaster smooths the image before detecting spots, using a smooth-by-diffusion algorithm. The Smooth parameter should be optimized to detect all real spots and split as many overlapping spots as possible.
2. Saliency: This parameter is a measure based on the spot curvature. It indicates how far a spot stands out with respect to its environment. Real spots generally have high saliency values whereas artifacts and background noise have small saliencies. Although the Saliency is an efficient quantity for filtering spots, it is also highly dependent on the images.
3. Min Area: After setting an appropriate Saliency to filter out all noise spots, there may still be noise in your gel that cannot be eliminated without suppressing real spots. This often happens with dust particles that consist of a few very dark pixels. Get rid of these artifacts by using the Min Area parameter. It eliminates spots that have an area smaller than the specified threshold (expressed in number of pixels). 2 3 4 5 4

5. 3 Step 1: 1 2 4 5 T1:Gel Scanning Audio Narration (if any)‏
Description of the action/ interactivity
Show the scanner connected to monitor, instruct user to open the scanner lid and place the stained gel to be scanned and close the lid This action should happen with user control. After the computer is turned on, log on to Windows. Locate and double-click the Scanner Control shortcut icon on the desktop.
Place the gel in the middle of the scanner platform, avoid air bubbles. Click on shortcut icon or user can start the Scanner Control software using the Start menu. The Scanner Control window appears 4 5 5

6. 3 Step 1: 1 2 4 5 T1:Gel Scanning Audio Narration (if any)‏
Description of the action/ interactivity
Display the Scanner Control window, select an existing template that contains the scan parameters you want to use, or manually select the parameters. To manually select the parameters, make sure Storage Phosphor/comassie blue is the selected scan acquisition mode. Choose whether you want best sensitivity or best resolution. Select the grid area or tray template, pixel size, sample orientation, and image analysis software. Click Scan. Type a file name and click Save.
User need to set the parameters before going for scanning. 4 5 6

7. Step 1: 1
T1:Gel Scanning 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Display the ImageQuant preview window. Animate scanning, display the scanned gel image with red spots, instruct user to cancel the scan and rescan with shorter amount of time. Display gel with green spots, instruct user to cancel the scan and set the expose time for longer time. Later display normal spots in gel and instruct user to save the gel.
During scanning, check the image in the ImageQuant preview window for saturation. Saturated pixels appear in red. If the image appears saturated, you might need to expose a clean screen to the sample for a shorter amount of time. If the image appears faint, you might need to expose a clean screen for a longer amount of time. If the image appears usable, continue with the next scan and save the image. Naming of gel must be proper with project name, type of sample etc. 4 5 7

8. Step 2: 1
T2:IMAGE Processing 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Double click on IMP7 icon, display the IMP7 analysis window. Choose: File:open:Workspace window, click the Project icon. In the Add Files window, browse the directory where the image file is located, select its name and click Open. Use the Shift or Ctrl keys to make multiple selections. Specify the staining. If the staining cannot be found in the list, you can type a new one. Click OK.
Create project, Load the control and treated gel images in folder for analysis. Once the gels are loaded cropping need to carried out. 4 5 8

9. 3 Step 2: 1 2 4 5 T2:IMAGE Processing
rotating icon
FLip icon 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Select the gel image to be rotated in the Image Pool sheet. Click the Free Rotate icon. A red grid must appear on the image. Click anywhere in the image and rotate the grid while holding the left mouse button. Animate user movement with the gel. You can also manually enter a rotation angle in the Rotation Tool window.
Images rotating need to be carried out to align all the gels in same orientation, image processing does not affect original image
files. The bold horizontal grid line plays the role of landmark to help you visualize the rotation. Flip icon is used when gel images are scanned in the wrong direction, you can Flip Horizontally or Flip Vertically to produce their correct mirror image. 4 5 9

10. Step 2: 1
T2:IMAGE Processing
CROP icon 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Instruct user to click on CROP icon. Click the Region tool and place the cursor at the top left position of the area you want to crop by holding shift button to crop equal region in all gels. Hold down the left mouse button and move the cursor to the bottom right position (a dashed box is displayed). Release the mouse button at the end point. Move a crop area by clicking inside the box and dragging it. Change the size of the area by dragging a corner or edge.
You can crop your gel images with the Crop tool. This creates new gels that only contain the selected area and removes the outer area. Once the gels are cropped the images are ready for the analysis. 4 5 10

11. 3 Step 3: 1 2 4 5 T3:SPOT Processing
Audio Narration (if any)‏
Description of the action/ interactivity
Display the gels to be detected by selecting a match set in the Workspace. Right-click and select Display in the contextual menu. (Gel must be displayed in the window). Choose View : Spots : Outlined to visualize the spot borders.
Select the gel images for spot detection. Choose Edit : Spots : Detect
Animate the steps with user interaction and control
Once gels have been added to a project and user can have a good look at them, gels are ready for spot detection. A spot delineates a small region in the gel where protein is present. This shape is automatically differentiated by a spot detection algorithm and quantified; its intensity, area and volume are computed. 4 5 11

12. 3 Step 3: 1 2 4 5 T3:SPOT Processing
Audio Narration (if any)‏
Description of the action/ interactivity
The Detect Spots window appears on-screen and the spots in the preview regions of the selected images are detected with the default parameters. Adjust the detection parameters, let user inserts the values to optimize Smooth, Min Area and Salency. If the user is satisfied with the preview, instruct to press OK, if not instruct to change the parameters.
To detect the spots you need to define the optimum spot detection parameters. The parameter smooth helps to detect all real spots and split the overlapping ones. Subsequently, Saliency and Min Area values helps out to filter the noise. 4 5 12

13. Zoom option
Step 3: 1
T3:SPOT Processing 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Soon after detection of spots, highlight the spots with “+” at the centre of a spot or make a boundary for the spot as shown in fig. let user have a control of enlarging the area within the gel by controlling Zoom option in the tool bar.
After spot detection, zoom each and every spots to check for a real spot. The software detects dusts, artifacts, which needs to removed from analysis. 4 5 13

14. Step 3: 1
T3:SPOT Processing 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Select the unwanted spots, for example: right click for options like: detect spot, delete spot, land mark. Let user select choice of his own. For detect spot: add a spot at selected position. For delete spot: remove the selected spot. For land mark: highlight the spot with red color.
User must detect dusts, artifacts from the detected spots. which needs to be removed from analysis. If user finds unwanted spot, he can delete it and if he finds spots exactly at the same position across the gels, such spots can be landmarked. Land mark of spots helps in matching. Once land marking is over, save the gels and import the gels into Matching folder. 4 5 14

15. Step 4: 1
T4:GEL matching 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Instruct user to open the gels from the Match set folder. Display the gel images in gel display window, with few spots in orange as landmarked. If user clicks on the landmarked spots in one image, similar spot in other gel at same location must get selected, if not user should move to the correct spot by dragging the landmark to correct spot than only, landmark will get number. Once all spots are selected from edit: spots: select all spots, user can click on option Match gel: match all gels. A small window display: matching in process… continued by Matching gels is finished: 407 matches have been created to matchsets.
Matching of gels must be carried out after landmarking. Matching algorithm first matches the landmarked spots, then matches the nearby spots. In case if less number of matches are produced, create few more landmarks and try again. Landmarking must be in such a way that it should cover entire gel area. 4 5 15

16. Step 4: 1
T4:GEL matching 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Display the gel images in gel display window, with few spots in orange as landmarked. Instruct user to click on option View: spots: show vector lines. Once user selects the option, display short lines from each spots. The lines must be of same size and in same direction.
Vector lines helps to check the matching process, how correctly the gels have matched with eachother. If the vector lines are of same length and in same direction, we say matching is perfect. If the vector lines are all of different sizes and in different direction, we say matching is not perfect. 4 5 16

17. Step 4: 1
T4:GEL matching 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Now user as the option to overlay the gels to check matching. Instruct user to option click View: overlay gels. once user selects the option, display gels in two different color and a overlay image showing mixture of both the image.
The overlay option helps user to check the profile of each gel, to detect matching pattern.
Once overlay is done, and user is satisfied with matching user can proceed with the data analysis if not he can again restart the matching. Now save the workspace and drag the images in to classes folder. 4 5 17

18. 3 Step 5: 1 2 4 5 T5: Statistical DATA analysis
Audio Narration (if any)‏
Description of the action/ interactivity
Now click open the gels from classes folder. Select Edit: all spots, right click and check the option for Spot ID display. Now select option Report: spot table, a table with values must appear at the bottom. Animate user control for above steps.
The overlay option helps user to check the profile of each gel, to detect matching pattern.
Once overlay is done, and user is satisfied with matching he can proceed with the data analysis if not he can again restart the matching. 4 5 18

19. Step 5: 1
T5: Statistical DATA analysis 2 3
Audio Narration (if any)‏
Description of the action/ interactivity
Now when user selects individual spots from the gel, spot information with values in the form of table must get displayed in the RHS with right click option and select spot information.
Spots across the gel can be selected and compared to know their pI, volume, intensity, MW etc. 4 5 19

20. 3 Step 5: 1 2 4 5 T5: Statistical DATA analysis
Audio Narration (if any)‏
Description of the action/ interactivity
The user should select one spot and right click to select the 3D view option. The protein spot must be redraw for 3D animation and can be rotated in any angle as the user moves the cursor.
The protein spots can be represented in the 3-D form with peak height denotes its intensity and can be rotated to view in different angles. This helps user to make a rough calculation for the fold difference expression of protein between the samples. To make accurate calculation for fold difference, user must do the statistical analysis for the data.. 4 5 20

21. 3 Step 5: 1 2 4 5 T5: Statistical DATA analysis
Audio Narration (if any)‏
Description of the action/ interactivity
The user should click on statistical gel analysis from Report: statistical data, so that the table containing statistical data of the spots can be viewed. Before this table view, provide a small window check box with options like Intensity, volume, area and slope for user selection and later on user selection display the above table view.
Click on the statistical analysis table to get statistical information of the spots. The data can be used to produce fold difference between the spots, to determine increased and decreased spots, to generate a histogram for distribution of spots and calculate t-test , ANOVA , other statistical analysis. 4 5 21

22. Instructions/ Working area
Slide 8-10
Slide 18-21
Slide 11-14
Slide 15-17
Slide1 - 4
Slide
Introduction
Tab 01
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Name of the section/stage
Animation area
Interaction:1
In slide-15: instruct user to do analysis, provide user with two gels, having a little different pattern. Provide two option for user to carry out analysis: without landmark and with landmark.
Instruct: once user does both the analysis display more of matches in analysis with landmark in comparison with without landmark.
Interactivity
area
Button 01
Button 02
Button 03
Instructions/ Working area
Credits 22

23. Questionnaire: APPENDIX 1 Question 1 Data analysis is done to
To see the spots
To pick out significant spot for characterization
To make the spots visible
To detect the type of protein
Answer:To pick out significant spot for characterization
Question 2
Statistical analysis table give information about
a) Proteome
b)Type of sample
c) Statistically insignificant spot for characterization
d) Statistically significant spot for characterization
Answer:d)Statistically significant spot for characterization 23

24. Questionnaire: APPENDIX 1 Question 3
Spot volume table give information about
a)% volume of the spot
b)Volume intensity of the gel
c)Volume intensity of the proteome spot
d)Volume intensity of the 3D peaks
Answer:c)Volume intensity of the proteome spot
Question 4
Landmark is set to
a) Align the gels
b) Align the gel
c) Align the BSA
d) See the proteome
Answer: a)Align the gels 24

25. a) Crop the gel to reduce the size
APPENDIX 2
Question 5
Cropping is done to
a) Crop the gel to reduce the size
b) Crop the spots to reduce the intensity
c) Crop the gels with the region containing maximum number of spots
d) Crop the gels with the region containing minimum number of spots
Answer: c)Crop the gels with the region containing maximum number of spots 25

26. Links for further reading
APPENDIX 2
Links for further reading
Books:
ImageMaster 2D Platinum 7.0 User Manual Edition AA.
Papers:
1. Cao et, al., Proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian
infectious bronchitis coronavirus, Proteome Science 2011, 9:11.
2. J. Xi et, al., Polyethylene glycol fractionation improved detection of
low-abundant proteins by two-dimensional electrophoresis
analysis of plant proteome, Phytochemistry 67 (2006) 2341–2348. 26

27. APPENDIX 3
Summary
Data analysis has to be carried out with more stringency to prevent the ignorance of any significant protein spots in the proteome analysis. This analysis forms the deciding factor for protein characterization by Mass spectrometry. Analysis and options will vary with the software but the procedure and the goal to be achieved is same in all the data analysis. 27