Flicker comparison of 2-Dimensional Electrophoretic Gels

Flicker Comparison of 2-Dimensional Electrophoretic Gels

Welcome To Flicker

http://open2dprot.sourceforge.net/Flicker

Introduction

Flicker is an open-source stand-alone computer program for visually comparing 2D gel images. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels are often difficult to compare because of rubber-sheet distortions. Flicker allows you to visually compare your gel images against each other or against those found in Internet databases. Many published Internet gels have a subset of spots identified which may make them useful to compare with your gels. Some of these Internet gels are active maps that you can click on a spot to inquire of its identity. You may be able to draw putative conclusions as to the identification of some spots in your gels that visually appear to be the same spots as in reference gels. The Flicker program integrates these various needs to help you try to make putative spot identifications. See the Quick start examples below for a short list of some of these methods.

Of course Flicker might be useful for comparing other types of images such as 2D-blots, 1D gels, serial microscope sections, time-lapse, microarrays, etc. - any type of image where there are distortions or intensity variation of the images, differing numbers of spots, and other systematic and procedural experimental differences. It can read black and white or color JPEG and GIF images, and black and white TIFF images (some un-banded color TIFF images are available).

The Flicker application is Java program that runs under the MS Windows, MacOS-X, Linux and Solaris operating systems. It is downloaded to and runs on your computer. Flicker has been made open source and was contributed to the Open2Dprot project. Both the executable binary as well as the source code is available.

The original version of Flicker was a Java applet that ran in your Web browser ([Lemkin97a]-[Lemkin97c], [Lemkin99], [Lemkin02]) and is available at http://www.ccrnp.ncifcrf.gov/flicker. However, being a Java applet, it had many limitations in its capabilities. To resolve these limitations it was converted in 2003 to a Java application by Peter Lemkin and Greg Thornwall with help from Jai Evans. Code was added from the open-source MicroArray Explorer program, http://maexplorer.sourceforge.net/, to implement some of the new features in Flicker. The new version of Flicker is much more flexible and makes it easier for you to compare your gels with each other or with reference gels on Internet databases. A limited measurement functionality is available to estimate spot or region quantification.

The new version is described in a book chapter Comparing 2-D Electrophoretic Gels Across Internet Databases, PF Lemkin, GC Thornwall, J Evans (2005) in "The Proteomics Handbook", JM Walker (Ed), Humana Press Inc, Totowa, NJ, pp 279-305.

Notice: some individuals have problems looking at flickering images
Some individuals have problems with flickering images. The Flicker program allows you to flicker-compare two images at flicker rates of 0.1 second/frame up to 3 seconds/frame (individually selected on a per-image basis). If you are bothered by flickering images, do not download this program or do not use the flicker image comparison option. However, the program can be used without flicker and may still be useful to zoom/dezoom images, adjust brightness/contrast, do image transforms, identify and label spots via comparison with SWISS-2DPROT images, etc. In this case, don't enable the flicker option.

Quick start examples

The following are brief sketches of several ways that Flicker could be used. There are many other ways of using Flicker, and these are detailed in the Reference Manual.

A. Compare two gels to find spot differences.

Open 2 gels to compare (your own or gels from the Internet).
Flicker align similar regions for the spot(s) of interest.
When they are aligned, you can see local spot differences between the gels.

B. Compare your gel against an Internet reference gel to try to putatively identify the spot.

Open 2 gels to compare (let one of them be an active reference gel).
Flicker align similar regions for the spot(s) of interest.
If one of the gels is an active reference gel, then you can click on it to putatively identify the protein.

C. Quantitate a list of spots. Flicker has a rudimentary spot list definition and quantification capability that might be used as follows:

Open 2 gels to compare.
Flicker align similar regions for the each of the spot(s) of interest.
Add spots of interest to spot lists (a separate list for each gel).
Click on corresponding spots in each gel and pair them using a common annotation id.
List the spots in the paired spot list (this can be generated as tab-delimited data for export to Excel).

D. Putatively identify a list of spots in your gel that are identified in an active reference gel by first identifying spots in the reference gel and then using them to identify corresponding spots in your gel.

Open 2 gels to compare (let one of them be an active reference gel).
Flicker align similar regions for each of the spot(s) of interest.
Add spots of interest to spot lists (a separate list for each gel).
Request Flicker to visit the active reference gel Web server and try to lookup the protein id (e.g, Swiss-PROT) for the spots you have defined in the active gel.
Then click on corresponding spots in your gel and then pair them using a common annotation id from the reference gel.
List the spots in the paired spot list (this can be generated as tab-delimited data for export to Excel).

Use of 2D-PAGE in modern proteomics

This approach may be useful for comparing similar protein samples created in different laboratories to help putatively identify or suggest possible protein spot identifications. The gels should be run under similar pH and molecular weight ranges if possible. Although available for over three decades, 2D polyacrylamide gel electrophoresis (2D-PAGE) is still routinely used [1] even considering the now common use of mass spectrometry [2-7] and recently protein arrays [8] for protein identification. If you have defined a list of spots in an active gel (such as one of the Swiss-2DPAGE gels) and you are connected to the Internet, you can request Flicker to lookup the annotation information (Swiss-Prot id and name) for each spot in the list. Then you can define a list of spots in your gel that correspond to spots in the annotated active reference gel, and then assign these protein identifications to your gel.

Recent advances, such as IEF "zoom" fractionation gels [9] that divide the protein sample by pH range or immunoaffinity subtraction with LC [5], greatly increase the resolution and numbers of spots able to be discriminated by subsequent 2D-gel electrophoresis. Another increasingly common image comparison technique uses 2 to 6 cyanine dyes using dye multiplexing to label multiple control and experimental samples run in the same gel such as DIGE [10] and scanned with very high resolution systems [11]. Multiple scans of the same gel using different color filters can then be color mapped to see the contributions of the different samples. This is useful if one has control over the experimental design when determining the reference gel, set of control gels, and experimental gels. However, it does not solve the problem of trying to putatively compare one's own sample against an Internet reference gel where they have identified protein spots.

Finding reference gels on the Internet

A number of 2D-gel image databases are available on the Web where some of the proteins are identified for various types of samples. Both WORLD-2DPAGE and 2D-HUNT on the SWISS-2DPAGE [12-15] server can be used to find Web URL addresses for a number of 2D protein gel databases. The Web site a large number of tissues with databases that include a wide range of human tissues, mouse tissues, E. coli, aribidopsis, dictyostelium, and yeast. You might also try a Google 2D-gel search.

Active 2D image maps in Internet 2D-gel databases

Some 2D-gel image Web databases have active maps (e.g., SWISS-2DPAGE) where you can click on a spot in an active gel image to identify the protein if it is in their database (see WORLD-2DPAGE). Clicking on a spot queries the associated Web server database to determine if the spot you pointed to is in that database. If it is, it then reports the protein identity of the spot with links to SWISS-PROT etc. Alternatively, you can have it report the ID and protein name in the reporting window or assign it to the selected spots annotation.

Using active maps with Flicker to putatively identify protein spots

We have integrated this capability in Flicker so that if you are viewing an active map (say from SWISS-2DPAGE), you can switch from Flicker mode to Clickable image database (DB) mode and then just click on the spot to query the database which then will bring up the specific protein annotation Web page from the associated active DB server (e.g., SWISS-2DPAGE) in a Web browser. This is most useful after you have flicker-aligned your gel with the reference gel map image. Comparing one's own experimental 2D gel image data with gel images of similar biological material from such Internet reference databases opens up the possibility of using the spots in these reference gels to suggest the putative identification of apparently corresponding spots in your gels. The image analysis method described here allows scientists to more easily collaborate and compare their gel image data over the Web.

Methods for 2D gel comparison

When two 2D gels are to be compared, simple techniques may not suffice. There are a several methods for comparing two gel images: 1) put the images side by side and visually compare them; or 2) slide one gel (autoradiograph or stained gel) over the other while back lighted; or 3) build a 2D gel quantitative computer database from both gels after scanning and quantitatively analyzing these gels using a 2D gel database system; 4) more recently dye multiplexing has been used to label different samples in the same gel. A variant if this is to warp two gels so they are the same geometry, then generate pseudo color images and look for differences by color differences. These methods may be impractical for many investigators since in the first case the physical gel or autoradiograph from another lab may not be locally available. The first method may work for very similar gels with only a few differences. The second method will work better for gels that are not so similar but that have local regions that are similar. The third method may be excessive if only a single visual comparison is needed because of the costs (labor and equipment) of building a multi-gel database solely to answer the question of whether one spot is probably the same spot in the two gels. The fourth method may have some problems if spot sizes vary for similar spots.

Flicker was developed to fill the need for a quick comparison of a researcher's gel image against one of these internet reference 2D-gels. We have also provided a limited quantification facility for manually measuring, annotating, a limited number of spots.

The Flicker Reference Manual describes the operation of the various commands. The current status of the program and the revision history is updated as changes occur in the software. You download the software to install it on your computer. Flicker is downloaded as a Zip file from the Files Mirror.

Please contact us with suggestions and comments. If you make interesting changes in the source code, please send us a copy and describe your changes so we can merge them in the released version.

Contact us Flicker is a contributed program available at Open2Dprot.sourceforge.net/Flicker
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