Computer analysis of 2DE gel images

Once proteins in a sample have been separated by two-dimensional gel electrophoresis (2-DE), the main remaining task is to analyze the resulting

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Effect of troglitazone on pancreatic islets proteins expression. Comparison of the expression level of the polypeptide PTM1 in 2-DE gels under four conditions: A) LC: lean control c57Bl/6 female mice (125 islets: 95% of the islets were small, 5% were large), B) LT: lean treated c57Bl/6

female mice for one week with 300 mg/kg of troglitazone (135 islets: 95% small, 5% large), C) OC: obese ob/ob controls c57bl/ 6 female mice (200 islets: 20% small, 80% large), and D) OT: obese ob/ob treated c57Bl/6 female mice for one week with 300 mgs/kg of troglitazone (200 islets: 40% small.60% large).

Effect of troglitazone on pancreatic islets proteins expression. Comparison of the expression level of the polypeptide PTM1 in 2-DE gels under four conditions: A) LC: lean control c57Bl/6 female mice (125 islets: 95% of the islets were small, 5% were large), B) LT: lean treated c57Bl/6

female mice for one week with 300 mg/kg of troglitazone (135 islets: 95% small, 5% large), C) OC: obese ob/ob controls c57bl/ 6 female mice (200 islets: 20% small, 80% large), and D) OT: obese ob/ob treated c57Bl/6 female mice for one week with 300 mgs/kg of troglitazone (200 islets: 40% small.60% large).

2-DE images and to extract biologically relevant information. As researchers in the Life Sciences do not use the 2-DE technology with only one goal in mind, there is obviously more than one way to consider the analysis of 2-DE images. In general, one may consider three main approaches that vary in the globality or specificity of the proteins under study:

1. The creation of proteome maps. This is an important step towards the discovery of a full proteome. It is the systematic analysis of all (or as many as possible) spots contained on one 2-DE image of a given sample, in order to identify the corresponding proteins (see Section 4.4). The 2-DE image is then annotated with the proteins' names as well as additional related data, and the resulting protein map is added to a two-dimensional electrophoresis database, such as the SWISS-2DPAGE (http://www.expasy.org/ch2d/) [20, 6], the HSC-2DPage [21] or the Siena-

2DPage [22] databases. Such databases, often available on-line on the Web, allow for complex queries in order to retrieve data related to proteins and their location on the 2-DE maps [23]. Figure 4.3 shows the annotated 2-DE image of Escherichia coli, the related 2-DE image as it can be retrieved from SWISS-2DPAGE, as well as one of the textual database entries (in this case Elongation factor TU P02990). Most 2-DE databases available on the Web follow the rules for federated databases [24], that allow, from each entry in the database, to follow active hypertext links to the corresponding entries in other proteomic or genomic databases, such as the SWISS-PROT protein sequence [25] or the EMBL nucleotide sequence [26] databases (Figure 4.4) (see also Section 4.5).

2. The monitoring of the expression of one or a few gene products over a set of 2-DE gels. This approach encompasses the pharmacological or toxic effects of compounds on a disease process, either over a given time span or under various compound concentrations, as well as, more generally, when one is interested to measure the variation of the expression of a protein over a series of 2-DE gels of a given sample. To achieve such measurements, a set of 2-DE images is considered, and the analysis focuses on quantitative variations limited to the proteins of interest.

3. The global investigation of the differential expression of proteins between 2-DE images of two or more populations. A typical example is the identification of polypeptides that correlate with disease states. In this case two sets of 2-DE gels from disease versus control are compared to identify which proteins are consistently differentially expressed between the two sets of images. Alternatively, one compares samples from two or more disease states or from any biological conditions whose differential analysis might be of interest. In general, this global approach thus entails the comparison of the spot intensities in two or more (often large) sets of 2-DE images, leading to the further exploration of a few to several tens of gene products that could be of potential biological importance, such as, typically, potential disease markers or therapeutic targets. In given situations, researchers may also be interested in the investigation of only one set of 2-DE images, in order to discover patterns or subsets of samples that could possibly be linked to specific biological conditions or disease states. In any case, each variant of this approach necessitates the use of powerful data analysis tools, such as statistical programs to identify the proteins that are differentially expressed. This part of proteome analysis is very often followed by the identification and partial characterization of the proteins that have been pinpointed, using the techniques described in Section 4.4.

The three approaches of 2-DE analysis described above differ mainly in the number of 2-DE images that are considered in one given study and in the kind of image or protein comparisons that are performed. In the first approach, typically one 2-DE image is considered at a time, while in the

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The entries in a federated 2-DE database like SWISS-2DPAGE contain active hyperlinks to cross-referenced databases, like SWISS-PROT and GeneBank.

third one, up to several thousands of 2-DE gels may be examined simultaneously, and thus the expression levels of sometimes several hundred thousands of proteins must be analyzed. This approach can only be carried out with the assistance of powerful 2-DE analysis software packages.

Continue reading here: Short history of 2DE analysis by computer

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