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Open Access Highly Accessed Research article

Normalization and expression changes in predefined sets of proteins using 2D gel electrophoresis: A proteomic study of L-DOPA induced dyskinesia in an animal model of Parkinson's disease using DIGE

Kim Kultima1*, Birger Scholz1, Henrik Alm1, Karl Sköld23, Marcus Svensson23, Alan R Crossman4, Erwan Bezard5, Per E Andrén23 and Ingrid Lönnstedt16

Author Affiliations

1 Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, BMC, Box 594, SE-75124 Uppsala, Sweden

2 Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Box 583, SE-75123 Uppsala, Sweden

3 Department of Pharmaceutical Biosciences, Uppsala University, BMC, Box 583, SE-75123 Uppsala, Sweden

4 Faculty of Life Sciences, The University of Manchester, UK

5 CNRS UMR 5543, University Victor Segalen, Bordeaux, France

6 Department of Mathematics, Uppsala University, Box 480, SE-75106 Uppsala, Sweden

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BMC Bioinformatics 2006, 7:475  doi:10.1186/1471-2105-7-475

Published: 26 October 2006



Two-Dimensional Difference In Gel Electrophoresis (2D-DIGE) is a powerful tool for measuring differences in protein expression between samples or conditions. However, to remove systematic variability within and between gels the data has to be normalized.

In this study we examined the ability of four existing and four novel normalization methods to remove systematic bias in data produced with 2D-DIGE. We also propose a modification of an existing method where the statistical framework determines whether a set of proteins shows an association with the predefined phenotypes of interest. This method was applied to our data generated from a monkey model (Macaca fascicularis) of Parkinson's disease.


Using 2D-DIGE we analysed the protein content of the striatum from 6 control and 21 MPTP-treated monkeys, with or without de novo or long-term L-DOPA administration.

There was an intensity and spatial bias in the data of all the gels examined in this study. Only two of the eight normalization methods evaluated ('2D loess+scale' and 'SC-2D+quantile') successfully removed both the intensity and spatial bias. In 'SC-2D+quantile' we extended the commonly used loess normalization method against dye bias in two-channel microarray systems to suit systems with three or more channels.

Further, by using the proposed method, Differential Expression in Predefined Proteins Sets (DEPPS), several sets of proteins associated with the priming effects of L-DOPA in the striatum in parkinsonian animals were identified. Three of these sets are proteins involved in energy metabolism and one set involved proteins which are part of the microtubule cytoskeleton.


Comparison of the different methods leads to a series of methodological recommendations for the normalization and the analysis of data, depending on the experimental design. Due to the nature of 2D-DIGE data we recommend that the p-values obtained in significance tests should be used as rankings only. Individual proteins may be interesting as such, but by studying sets of proteins the interpretation of the results are probably more accurate and biologically informative.