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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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2006 (English)In: BMC Bioinformatics, ISSN 1471-2105, Vol. 7, 475- p.Article in journal (Refereed) Published
Abstract [en]

Background: 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.

Results: 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.

Conclusion: 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.

Place, publisher, year, edition, pages
2006. Vol. 7, 475- p.
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-95980DOI: 10.1186/1471-2105-7-475ISI: 000241904700001PubMedID: 17067368OAI: oai:DiVA.org:uu-95980DiVA: diva2:170380
Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2011-05-12Bibliographically approved
In thesis
1. Transcriptomics and Proteomics Applied to Developmental Toxicology
Open this publication in new window or tab >>Transcriptomics and Proteomics Applied to Developmental Toxicology
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Developmental toxicology is an important part of preclinical drug toxicology as well as environmental toxicology. Assessing reproductive and developmental toxicity is especially expensive and time demanding, since at least two generations of animals are needed in the tests. In light of this there is a great need for alternative test methods in many areas of developmental toxicity testing.

The complete set of RNA transcripts in any given organism is called the transcriptome. Proteomics refers to the study of the proteins in a given organism or cell population. The work of this thesis has focused on the use of high throughput screening methods in transcriptomics and proteomics to search for molecular markers of developmental toxicity.

We have studied the global gene expression effects of the developmentally toxic substance valproic acid (VPA) using microarray technology. Several genes were found that display the same gene expression pattern in vivo using mouse embryos as the pattern seen in vitro using the embryocarcinoma cell line P19. Based on these observations, the gene Gja1 was suggested as one potential molecular marker of VPA induced developmental toxicity and potential marker of histone deacetylase (HDAC) inhibition in vitro.

Using 2D-DIGE technology, which measures relative protein abundances, the effect of neonatal exposure to the flame retardant PBDE-99 was studied in mouse brain (cortex, hippocampus and striatum) 24 hr after exposure. Differentially expressed proteins in the cortex and the striatum indicate that PBDE-99 may alter neurite outgrowth.

Finally, we have suggested several improvements in the use of the 2D-DIGE technology. Novel methods for normalizing data were presented, with several advantages compared to existing methods. We have presented a method named DEPPS that makes use of all identified proteins in a dataset to make comprehensive remarks about biological processes affected.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 50 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 58
Toxicology, 2D-DIGE, Valproic acid, PBDE-99, Flame retardant, Neural tube defects, Microarray, In vivo, In vitro, Biomarker, Brain growth spurt, Normalization, Mouse embryo, Toxicogenomics, Toxikologi
urn:nbn:se:uu:diva-7921 (URN)978-91-554-6914-6 (ISBN)
Public defence
2007-06-08, B42, BMC, Husargatan 3, Uppsala, 09:15
Available from: 2007-05-15 Created: 2007-05-15Bibliographically approved

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Kultima, KimScholz, BirgerAlm, HenrikSköld, KarlSvensson, MarcusAndrén, Per E.
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