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Proteomics in biomarker research: Insights into the effects of aging and environment on biological systems
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteomics is the global analysis of proteins that covers a broad range of technologies aimed at determining the identity and quantity of proteins expressed in the cell, their three-dimensional structure and interaction partners. In contrast to genome, proteome reflects more accurately on the dynamic state of the cell, tissue, or an organism. Therefore much is expected from proteomics to yield better disease markers for early diagnosis and therapy monitoring, as well as biomarkers that would indicate environmental exposure or provide prediction of biological age.

In this thesis, I have developed and applied robust and sensitive subproteomic approaches to study the effect of aging as well as and environmental pollution using different animal models.

In the first part, a high-throughput proteomic method based on liquid chromatography coupled to 2-dimensional gel electrophoresis (LC/2-DE) was developed. The usefulness of this method has been demonstrated by applying it to the assessment of marine pollution in a field experiment.

Next, I have utilized this subproteomic approach to study the effect of aging in mouse kidney of both genders. As a result, a protein expression signature of aging kidney was obtained, revealing gender-dependent alterations in proteome profiles of aging mouse kidney.

In order to further reduce the dynamic range of protein expression and increase the sensitivity of proteomic analysis, I have applied a shotgun mass spectrometry-based proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to study age-related differences in peroxisome-enriched fractions from mouse liver. Only eight proteins showed statistically significant difference in expression (p<0.05) with moderate folds. This study indicates that age-depended changes in the liver proteome are minimal, suggesting that its proteome is efficiently maintained until certain age.

Finally, in the context of aging studies and the role of peroxisomes in aging, I have tested the utility of cell-penetrating peptides (CPPs) as agents for protein delivery into acatalasemic peroxisomes using yeast as a model. The results obtained suggest that CPPs may be suitable for the delivery of antioxidants to peroxisomes and in future could provide a tool for the protein therapy of age-related diseases.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2011. , 63 p.
Keyword [en]
proteomics, biomarker, peroxisomes, 2-dimensional gel electrophoresis (2-DE), mass spectrometry (MS), aging, marine pollution
National Category
Biochemistry and Molecular Biology Environmental Sciences Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-56483ISBN: 978-91-7447-209-7OAI: oai:DiVA.org:su-56483DiVA: diva2:411567
Public defence
2011-05-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following publications were unpublished and had a status as follows: Paper 3: Submitted, Paper 4: Submitted.Available from: 2011-05-05 Created: 2011-04-18 Last updated: 2011-05-10Bibliographically approved
List of papers
1. Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis
Open this publication in new window or tab >>Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis
2007 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 6, 2094-2104 p.Article in journal (Refereed) Published
Abstract [en]

Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel “blue mussel” (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI−MS/MS), and they are involved in β-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.

Keyword
Amino Acid Sequence, Animals, Cell Fractionation, Chromatography; Ion Exchange/methods, Electrophoresis; Gel; Two-Dimensional/methods, Molecular Sequence Data, Mytilus edulis/*chemistry, Oceans and Seas, Proteins/*analysis, Proteomics/*methods, Spectrometry; Mass; Electrospray Ionization, Water Pollution; Chemical
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-11250 (URN)10.1021/pr060689s (DOI)000246893500004 ()
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2011-04-20Bibliographically approved
2. Proteomic study on gender differences in aging kidney of mice
Open this publication in new window or tab >>Proteomic study on gender differences in aging kidney of mice
2009 (English)In: Proteome Science, ISSN 1477-5956, Vol. 7, no 16Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

RESULTS: This proteomic analysis detected age-related changes in protein expression in 55 protein-spots, corresponding to 22 spots in males and 33 spots in females. We found a protein expression signature (PES) of aging composed by 8 spots, common for both genders. The identified proteins indicated increases in oxidative and proteolytic proteins and decreases in glycolytic proteins, and antioxidant enzymes.

CONCLUSION: Our results provide insights into the gender differences associated to the decline of kidney function in aging. Thus, we show that proteomics can provide valuable information on age-related changes in expression levels of proteins and related modifications. This pilot study is still far from providing candidates for aging-biomarkers. However, we suggest that the analysis of these proteins could suggest mechanisms of cellular aging in kidney, and improve the kidney selection for transplantation.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-34703 (URN)10.1186/1477-5956-7-16 (DOI)000266424100001 ()19358702 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2011-04-20Bibliographically approved
3. Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC-MS/MS
Open this publication in new window or tab >>Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC-MS/MS
(English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376XArticle in journal (Refereed) Submitted
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56481 (URN)
Available from: 2011-04-18 Created: 2011-04-18 Last updated: 2011-04-20Bibliographically approved
4. Delivering catalase to yeast peroxisomes using cell-penetrating peptides
Open this publication in new window or tab >>Delivering catalase to yeast peroxisomes using cell-penetrating peptides
(English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658Article in journal (Refereed) Submitted
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-56482 (URN)
Available from: 2011-04-18 Created: 2011-04-18 Last updated: 2015-04-21

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