Research Article |
Open Access |
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AC2DGel: Analysis and Comparison of 2D Gels |
Amit Kush and G.P.S. Raghava * |
Institute of Microbial Technology, Sector 39-A, Chandigarh, PIN-160036, INDIA |
| *Corresponding author: |
Dr. G. P. S. Raghava, Scientist, Institute of Microbial Technology, Sector 39-A, Chandigarh, PIN-160036, INDIA,
Fax : +91-172-2690632 or 2690585,
Tel : +91-172-2690557 or 2690225,
E-mail : raghava@imtech.res.in,
Web : http://imtech.res.in/raghava |
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| Received March 02, 2008; Accepted April 15, 2008; Published April 22, 2008 |
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Citation: Kush A, Raghava GPS (2008) AC2DGel: Analysis and Comparison of 2D Gels. J Proteomics Bioinform 1: 043-046. doi:10.4172/jpb.1000008 |
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Copyright: © 2008 Kush A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited. |
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Two-dimensional gel electrophoresis can retrieve information regarding thousands of different proteins from a crude protein
sample. One of the major challenges in field of proteomics is to extract maximum information from 2D gels. In this study, we
developed a web server for the analysis and comparison of 2D gels, which consists of three major modules. The first module
allows the analysis of gels on the basis of molecular weight and pH. This module assists in calculating molecular weight and pH
of a protein by clicking on corresponding spot at 2D gel image. The second module allows the comparison of two gels and
presents the result as a superimposed image where spots/proteins on two gels can be examined. The useful feature of this module
is that it allows the comparison of whole gel images or user specified areas or spots of gels. Besides this, it also allows zooming and
other image transformations such as brightness and contract enhancement. The third module is an interface to the database of
2-D gel images maintained locally. The database consists of information about more than 3500 well annotated 2-D gel images
obtained from public databases and literature. The server allows searching of gels from the database by keyword. Web server
AC2Dgel is available for public from http://www1.imtech.res.in/raghava/ac2dgel/. |
Introduction |
In the post-genomic era, proteomics has emerged as a new and
powerful tool for the analysis and annotation of protein data
( Ong
and Pandey, 2001). Despite of many pitfalls, 2-D gel electrophoresis
( O’Farrell, 1975)) has proved to be the most
comprehensive technique for the analysis of proteome. It is popular
in proteomics due to its ability to separate proteins on the
basis of their molecular weight and pH gradient. The two-dimensional
gel electrophoresis is also useful in identifying the differential
expression of genes under different conditions. In 2-D gels
a complex pattern of protein spots is usually observed due to
unique behavior of proteins when they are separated on the basis
of pH and molecular weight simultaneously. A single 2-D gel
have large number of protein spots differing slightly in their concentration
which make the visual analysis of these gels very time
consuming and cumbersome task. So, there is a great demand for
computation tool for the automated analysis of gels. This motivated
computer biologists to develop tools for the analysis of
virtual 2-D gel images.
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In the past, several computation packages have been developed
for the analysis of 2-D gels such as Melanie, Delta 2D and Z3.
These packages provide number of options for manipulation and
analysis of gels. Melanie (Appel et al., 1997) is a comprehensive
package for the analysis of multiple 2-D gel images. This
software facilitates analysis of protein spots in virtual 2-D gel
images. Similarly, Delta 2D and Z3 are other packages for the
manipulation and comparison of virtual 2-D gel images. The
major limitation of these packages is that they are available only
for Microsoft Windows and require large memory for processing.
These requirements restrict their usage on the personal computers.
In order to overcome the limitations of these packages,
many dynamic web servers have been developed for the manipulation
of images. The Flicker and Carol are two major online
web servers for the analysis of 2-D gel images. The Flicker compares
the two gel images using flicker method, in which two
aligned gel images are shown simultaneously in the same visual
space one by one (Lemkin, 1997). On the other hand, Carol
web server analyzes the gel images on the basis of pattern matching
algorithm (Pleissner, 1999). These servers run with high
speed internet connectivity and Java enabled browsers. Thus, all the packages available for 2-D gel analysis have few merits and
few demerits. In order to complement the existing 2-D gel analysis
packages, we developed a new web server. This server allows
the comparison and analysis of 2-D gel images. The server also
provides an interface to a database of locally maintained 2-D
gels. The server is platform independent and freely available for
academicians from http://www2.imtech.res.in/raghava/ac2dgel/ . |
Description of the Web Server |
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The server has been systematically developed by constructing
three modules for analysis and comparison of 2-D gel
images. Following is the brief description of modules. |
Analysis of Gels |
| This module allows annotation of proteins based on their
molecular weight and pH. The user can compute the molecular
weight and pH of protein spots on gel by clicking on the corresponding
spot. In order to compute molecular weight and pH of
protein, user need to specify the molecular weight and pH with
their scales corresponding to standard or known markers of spots
(Figure 1a). The user can upload a virtual image of gel in any
standard format. The module uses the linear square curve fitting
technique to fit markers data (e.g. molecular weight vs distance
of spots from bottom and pH vs distance of spots from left) for
computing linear equations, which serves as the internal calibration
for determining the molecular weight and pH of the experimental
proteins on the gel. The module calculates pH and molecular
weight using the linear equations derived from marker
data corresponding to the spot on gel by clicking on it (Raghava,
1994) (Figure 1b). Thus, user can choose any point on the
image to calculate the molecular weight and pH of protein of
specified location. The tool also allows to further search the protein
of similar molecular weight and pH from the database as
chosen by user. The list of all proteins of related molecular weight
can be displayed in the result. The user can choose any gel from
the result to compare it with his gel of interest. In summary, this
module allows assignment of pH and molecular weight of protein
of interest. The combination of this tool with gel comparison
module allows functional annotation of protein. |
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Figure 1: Demonstration of application of ‘Gel Analysis’ module; a) example submission form to input information about standard
markers and b) demonstration of calculation of molecular weight and pH by clicking on any spot.
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Comparison of Gels |
| This module facilitates the comparison of two images corresponding
to 2-D gels. One of the advantages of this module is that it
allows user to upload/get images to be compared, directly from
various resources. That includes uploading of images from local
disk and downloading from Internet by providing the URL of
image. It also allows the comparison of images stored in local
databases maintained by user. The method can accept 2-D gel
images in any standard image format such as Joint Photographic
Experts Group (jpeg), Graphics Interchange Format (gif) and Tag
Interchange File Format (tiff). All the input images from different
sources are first converted to a uniform format using the ‘convert’
subroutine of ImageMagick library. The images are then
processed through ‘gifinter’ function of the giflib library to convert
the interlaced images to non-interlaced. These images are
finally converted to gray scale tiff images for further manipulation.
This is done using functions of giflib and tiflib libraries
respectively for further manipulation of images. After processing
of images, server presents two images as shown in Figure 2a.
The user can perform the transformation of these images in term
of varying contrast or brightness of both images. This is carried
out using the ‘histex’ digital image manipulation tools (Seul
et al., 2000) and ‘convert’ subroutine. The images can then be
compared as a whole or the location of any specific spot or protein
to be compared can be specified. The specified area of image
is obtained using ‘gifclip’ function of giflib library for the
comparison. In last, the images are compared by inverting one of
the two images using the ‘imgarith’ function (Seul et al., 2000)
and superimposing it over the first one. The resultant image
(figure
2b) is zoomed up using the ‘xscale’ function (Seul et al.,
2000) and displays gray and white areas where gray specifies
areas common in between two images and white specifies the
areas, which differ in two images. This module can be used to
identify the change in protein profile under various psychological
conditions. |
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Figure 2: Example displays obtained from server which demonstrate its functions; (a) presents the images after processing, allowing
zooming of image and applying transformations and (b) the resultant superimposed image.
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Module for Data Storage and Retrieval |
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This module has been developed as an interface for the
database of 2-D gels. The database has more than 3500 well
annotated 2-D gels. These images were obtained from various
databases and literature, which are available free for academic
users. The module allows the keyword search on fields of
database such as accession number, master name or molecular
weight. It displays the complete information about gels like
molecular weight, pH, SWISS-PROT accession number of the
2-D gels that matched to users query. The results also display
an annotation virtual 2-D image and the information regarding
the source information and source organelle from which the gel
is generated. |
AC2DGel Description |
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The server has been launched on World Wide Web by
implementing the Apache web server (http://www.apache.org/)
on a Red Hat Linux 7.2 operating system (http://www.redhat.com/ ). The database is maintained locally using PostgreSQL (http://
www.postgresql.org/), one of the most advanced open source
database system available. The algorithms are implemented as
CGI scripts in Perl (http://www.perl.com/) language. Several inbetween
calculations and validations in the HTML forms have
been worked out using Javascripts. Several image analysis libraries
such as libtiff (http://www.libtiff.org/), giflib and
ImageMagick (http://www.imagemagick.org/) are used for image
handling and manipulation. In addition to these, some advanced
digital image manipulation and analysis tools (Seul et
al., 2000) have been used. These tools have been utilized for determining
the image size, conversion of images among different
formats, image clipping, image scaling, image superimposition,
determining intensity histogram, contrast enhancement and brightness
control. The information of 2-D gel images stored locally
has been drawn mainly from SWISS-PROT database (http://
www.expasy.ch/) Gasteiger et al., 2001; O’Donovan et al.,
2002; Junker et al., 1994) |
Acknowledgement |
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The authors are thankful to Ms. Amita Bansal and Ms.
Manjeet Kaur for initiating this project and developing various subroutines. We are also thankful to Mr. Manoj Bhasin for the
critical proofreading of the manuscript and his valuable suggestions. |
References |
-
Appel RD, Vargas JR, Palagi PM, Walther D, Hochstrasser DF
(1997) Melanie II—A third-generation software package for
analysis of two-dimensional electrophoresis images: II. Algorithms.
Electrophoresis 18: 2735-48. » CrossRef » PubMed » Google Scholar
-
Gasteiger E, Jung E, Bairoch A (2001) SWISS-PROT: connecting
biomolecular knowledge via a protein database. Curr Issues
Mol Biol 3: 47-55. » CrossRef » PubMed »
Google Scholar
-
Junker VL, Apweiler R, Bairoch A (1999) Representation of
functional information in the SWISS-PROT data bank.
Bioinformatics 15: 1066-1007. » CrossRef » PubMed »
Google Scholar
-
Lemkin PF (1997) Comparing Two-Dimensional gels across
the Internet. Electrophoresis 18: 461-470. » CrossRef » PubMed » Google Scholar
-
O Farrell PH (1975) High Resolution Two-Dimensional Electrophoresis
of Proteins. The Journal of Biological Chemistry 250:
4007-4021. » CrossRef » PubMed » Google Scholar
-
O Donovan C, Martin MJ, Gattiker A, Gasteiger E, Bairoch A,
et al. (2002) High-quality protein knowledge resource:
SWISS-PROT and TrEMBL. Brief Bioinform 3: 275-84. » CrossRef » PubMed » Google Scholar
-
Ong SE and Pandey A (2001) An evaluation of the use of
two-dimensional gel electrophoresis in proteomics. Biomolecular
Engineering 18: 195-205. » CrossRef » PubMed » Google Scholar
-
Pleissner KP, Hoffmann F, Kriegel K, Wenk C, Wegner S,
et al. (1999) New algorithmic
approaches to protein spot detection and pattern matching in twodimensional
electrophoresis gel databases. Electrophoresis 20:
755-65. » CrossRef » PubMed » Google Scholar
-
Raghava GPS (1994) Improved estimation of DNA fragment
lengths from gel electrophoresis. Biotechniques 17: 100-104. » Google Scholar
-
Seul M, O’Gorman L and Sammon MJ (2000) Practical
Algorithms for Image Analysis.
» CrossRef » Google Scholar
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