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The state of the art in the analysis of two-dimensional gel electrophoresis images.

Berth M, Moser FM, Kolbe M, Bernhardt J - Appl. Microbiol. Biotechnol. (2007)

Bottom Line: Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments.Challenges for analysis software as well as good practices are highlighted.We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.

View Article: PubMed Central - PubMed

Affiliation: DECODON GmbH, Rathenau-Strasse 49a, 17489 Greifswald, Germany.

ABSTRACT
Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.

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Scatter plot (a) of logarithmic spot quantities on two gels from different samples. Spots were normalized based on total spot quantity. b The quantile–quantile plot (QQ plot) of the same data. Spots are sorted by quantity separately on each gel; spots of corresponding ranks are plotted. The QQ plot makes it easier to compare the spot volume distributions; in an ideal experiment, all points would lie on the diagonal line. The diagram shows that the quantity distributions on both gels are nearly equal, indicating a successful normalization
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Fig12: Scatter plot (a) of logarithmic spot quantities on two gels from different samples. Spots were normalized based on total spot quantity. b The quantile–quantile plot (QQ plot) of the same data. Spots are sorted by quantity separately on each gel; spots of corresponding ranks are plotted. The QQ plot makes it easier to compare the spot volume distributions; in an ideal experiment, all points would lie on the diagonal line. The diagram shows that the quantity distributions on both gels are nearly equal, indicating a successful normalization

Mentions: Normalization procedures aim to mitigate systematic differences between images. Such variation can occur in protein loading, imaging exposure times, and dye/staining efficiency. Normalization starts with raw spot volumes and relates them to spot volumes on the same or on other gels. The simplest common procedure is to normalize for total volumes. The corresponding normalization rule is to set the total spot quantity to 100% on each gel image. For every spot, its proportion of total quantity is then computed. This rule results from the assumption that there is the same total protein amount on all gels. With this procedure, errors such as different protein loads, differences in staining times, scanner exposure time, or detector sensitivity can be compensated. For some instances, the total spot intensity is distorted by one or a few very strong spots leading to a skewing in the scatter plot (Fig. 12a). A countermeasure is to exclude some of the strongest spots (e.g., the 10% strongest spots) from the calculation of total spot intensity, i.e., removal of outliers.Fig. 12


The state of the art in the analysis of two-dimensional gel electrophoresis images.

Berth M, Moser FM, Kolbe M, Bernhardt J - Appl. Microbiol. Biotechnol. (2007)

Scatter plot (a) of logarithmic spot quantities on two gels from different samples. Spots were normalized based on total spot quantity. b The quantile–quantile plot (QQ plot) of the same data. Spots are sorted by quantity separately on each gel; spots of corresponding ranks are plotted. The QQ plot makes it easier to compare the spot volume distributions; in an ideal experiment, all points would lie on the diagonal line. The diagram shows that the quantity distributions on both gels are nearly equal, indicating a successful normalization
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279157&req=5

Fig12: Scatter plot (a) of logarithmic spot quantities on two gels from different samples. Spots were normalized based on total spot quantity. b The quantile–quantile plot (QQ plot) of the same data. Spots are sorted by quantity separately on each gel; spots of corresponding ranks are plotted. The QQ plot makes it easier to compare the spot volume distributions; in an ideal experiment, all points would lie on the diagonal line. The diagram shows that the quantity distributions on both gels are nearly equal, indicating a successful normalization
Mentions: Normalization procedures aim to mitigate systematic differences between images. Such variation can occur in protein loading, imaging exposure times, and dye/staining efficiency. Normalization starts with raw spot volumes and relates them to spot volumes on the same or on other gels. The simplest common procedure is to normalize for total volumes. The corresponding normalization rule is to set the total spot quantity to 100% on each gel image. For every spot, its proportion of total quantity is then computed. This rule results from the assumption that there is the same total protein amount on all gels. With this procedure, errors such as different protein loads, differences in staining times, scanner exposure time, or detector sensitivity can be compensated. For some instances, the total spot intensity is distorted by one or a few very strong spots leading to a skewing in the scatter plot (Fig. 12a). A countermeasure is to exclude some of the strongest spots (e.g., the 10% strongest spots) from the calculation of total spot intensity, i.e., removal of outliers.Fig. 12

Bottom Line: Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments.Challenges for analysis software as well as good practices are highlighted.We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.

View Article: PubMed Central - PubMed

Affiliation: DECODON GmbH, Rathenau-Strasse 49a, 17489 Greifswald, Germany.

ABSTRACT
Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.

Show MeSH
Related in: MedlinePlus