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Development of Methods for Protein and Peptide Analysis Applied in Neuroscience Utilizing Mass Spectrometry
Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the utilization of the matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and electrospray ionization (ESI) MS techniques for analysis of complex brain tissue samples.

Direct molecular profiling of biological samples using MALDI MS is a powerful tool for identifying phenotypic markers. MALDI MS-profiling of proteins and peptides directly on brain tissue sections was used for the first time to study experimental models of Parkinson’s disease (PD). The mass spectrometer was used to map the peptide and protein expression directly on 12 µm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein and peptide expression profile differences were found in the dopamine denervated brains when compared to the corresponding controls, for example, calmodulin, cytochrome c, cytochrome c oxidase, and the neuroimmunophilin protein FKBP-12. The increased expression of FKBP-12 from the profiling experiments was supported by mRNA expression analysis and two-dimensional gel electrophoresis separation analysis. Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar PD. This study showed for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted PD brain. The strength of the MALDI MS-profiling technique is that a minimum of sample handling and manipulation is necessary pre-analysis. This ensures preservation of the spatial localization of the biomolecules in the tissue section.

Biological liquid samples often contain high amounts of salt that is non-compatible with the ESI MS technique. A nano-flow capillary liquid chromatography (nanoLC) system coupled on-line with ESI-MS was used to study the metabolism of the peptide LVV-hemorphin-7 in the brain and blood using in vivo microdialysis. The microdialysis technique provides capabilities for very precise sampling in specific brain regions. The combination of on-line desalting and pre-concentration by nanoLC with ESI MS is a powerful tool to detect minute concentration of metabolic fragments and endogenous biomolecules.

The utilization of mass spectrometry in neuroscience applications provides a uniquely advantageous tool for the analysis of complex biochemical events that underlie the pathological symptoms expressed in different disease states. Furthermore, the MALDI-MS profiling technique shows great potential for the future with regards to proteome analysis and drug discovery.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 41
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 318
Keywords [en]
Analytical chemistry, Mass spectrometry, Parkinson’s disease, Metabolism, Profiling Mass Spectrometry, Proteomics
Keywords [sv]
Analytisk kemi
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-4685ISBN: 91-554-6104-2 (print)OAI: oai:DiVA.org:uu-4685DiVA, id: diva2:165489
Public defence
2004-12-01, B:41, BMC, 13:15
Opponent
Supervisors
Available from: 2004-11-09 Created: 2004-11-09Bibliographically approved
List of papers
1. In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry
Open this publication in new window or tab >>In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry
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2003 In: Rapid Communications In Mass Spectrometry, Vol. 17, p. 838–844-Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92417 (URN)
Available from: 2004-11-09 Created: 2004-11-09Bibliographically approved
2. Molecular Profiling of Experimental Parkinson’s Disease: Direct Analysis of Peptides and Proteins on Brain Tissue Sections by MALDI Mass Spectrometry
Open this publication in new window or tab >>Molecular Profiling of Experimental Parkinson’s Disease: Direct Analysis of Peptides and Proteins on Brain Tissue Sections by MALDI Mass Spectrometry
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2004 In: Journal of Proteome Research, Vol. 3, p. 289- 295Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92418 (URN)
Available from: 2004-11-09 Created: 2004-11-09Bibliographically approved
3. Increased Levels of Ubiquitin in the 6-OHDA-Lesioned Striatum of Rats
Open this publication in new window or tab >>Increased Levels of Ubiquitin in the 6-OHDA-Lesioned Striatum of Rats
2005 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 223-226Article in journal (Refereed) Published
Abstract [en]

Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92419 (URN)10.1021/pr049836h (DOI)15822896 (PubMedID)
Available from: 2004-11-09 Created: 2004-11-09 Last updated: 2017-12-14Bibliographically approved
4. Increased striatal mRNA transcription and active protein expression of the immunophilin FKBP-12 in experimental Parkinson’s disease models
Open this publication in new window or tab >>Increased striatal mRNA transcription and active protein expression of the immunophilin FKBP-12 in experimental Parkinson’s disease models
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-92420 (URN)
Available from: 2004-11-09 Created: 2004-11-09 Last updated: 2010-01-13Bibliographically approved

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