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Neuropeptidomics – Methods and Applications
Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The sequencing of genomes has caused a growing demand for functional analysis of gene products. This research field named proteomics is derived from the term proteome, which by analogy to genome is defined as all proteins expressed by a cell or a tissue. Proteomics is however methodologically restricted to the analysis of proteins with higher molecular weights. The development of a technology which includes peptides with low molecular weight and small proteins is needed, since peptides play a central role in many biological processes.

To study endogenous peptides and hormones, the peptidome, an improved method comprising rapid deactivation in combination with nano-flow liquid chromatography (LC) and mass spectrometry (MS) was developed. The method has been used to investigate endogenous peptides in brains of mouse and rat. Several novel peptides have been discovered together with known neuropeptides.

To elucidate the post mortem time influence on peptides and proteins, a time course study was performed using peptidomics and proteomics technologies. Already after three minutes a substantial amount of protein fragments emerged in the peptidomics study and some endogenous peptides were drastically reduced with increasing post mortem time. Of about 1500 proteins investigated, 53 were found to be significantly changed at 10 minutes post mortem as compared to control. Moreover, using western blot the level of MAPK phosphorylation was shown to decrease by 95% in the 10 minutes post mortem sample.

A database, SwePep (a repository of endogenous peptides, hormones and small proteins), was constructed to facilitate identification using MS. The database also contains additional information concerning the peptides such as physical properties. A method for analysis of LC-MS data, including scanning for, and further profiling of, biologically significant peptides was developed. We show that peptides present in different amounts in groups of samples can be automatically detected.

The peptidome approach was used to investigate levels of peptides in two animal models of Parkinson’s disease. PEP-19, was found to be significantly decreased in the striatum of MPTP lesioned parkinsonian mice. The localization and expression was further investigated by imaging MALDI MS and by in situ hybridization. The brain peptidome of reserpine treated mice was investigated and displayed a number of significantly altered peptides. This thesis demonstrates that the peptidomics approach allows for the study of complex biochemical processes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2006. , p. 57
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 42
Keywords [en]
Pharmaceutical pharmacology, mass spectrometry, proteomics, peptidomics, neuropeptide, bioinformatics
Keywords [sv]
Farmaceutisk farmakologi
Identifiers
URN: urn:nbn:se:uu:diva-7276ISBN: 91-554-6717-2 (print)OAI: oai:DiVA.org:uu-7276DiVA, id: diva2:169230
Public defence
2006-12-08, B42, BMC, Husargatan 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2006-11-17 Created: 2006-11-17Bibliographically approved
List of papers
1. A neuroproteomic approach to targeting neuropeptides in the brain.
Open this publication in new window or tab >>A neuroproteomic approach to targeting neuropeptides in the brain.
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2002 In: Proteomics, Vol. 2, no 4, p. 447-454Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-95132 (URN)
Available from: 2006-11-17 Created: 2006-11-17Bibliographically approved
2. Peptidomics-Based Discovery of Novel Neuropeptides.
Open this publication in new window or tab >>Peptidomics-Based Discovery of Novel Neuropeptides.
2003 In: J Proteome Res., Vol. 2, no 2, p. 213-219Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-95133 (URN)
Available from: 2006-11-17 Created: 2006-11-17Bibliographically approved
3. The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin (2-20) and peptides as sample quality indicators.
Open this publication in new window or tab >>The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin (2-20) and peptides as sample quality indicators.
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2007 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 24, p. 4445-4456Article in journal (Refereed) Published
Abstract [en]

Comparisons of transcriptional and translational expression in normal and abnormal states are important to reach an understanding of pathogenesis and pathophysiology. Maintaining the biochemical, molecular, and structural sample integrity is essential for correct sample comparisons. We demonstrate that both proteins and neuropeptides, including their PTMs, are subjected to massive degradation in the brain already 1 min postmortem. Further, markers for determining the integrity and status of a biological sample were identified. The protein fragment stathmin 2-20 correlated well with the general level of postmortem degradation and may serve as a sample quality indicator for future work, both in animal and human postmortem brains. Finally, a novel method for preventing degradation of proteins and peptides in postmortem tissue is presented using rapid and uniform conductive heat transfer on tissue prior to the actual sample preparation procedures, which enables the relatively low-abundant neuropeptides to remain intact, minimizes degradation of proteins by proteolysis, and conserves the PTMs of the neuropeptides.

Keywords
Phosphorylation, Protein, Peptides, Proteomics, Encephalon
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-95134 (URN)10.1002/pmic.200700142 (DOI)000252237000005 ()18072205 (PubMedID)
Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2018-01-13Bibliographically approved
4. SwePep – A database designed for endogenous peptides and mass spectrometry
Open this publication in new window or tab >>SwePep – A database designed for endogenous peptides and mass spectrometry
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2006 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 998-1005Article in journal (Refereed) Published
Abstract [en]

A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-95135 (URN)10.1074/mcp.M500401-MCP200 (DOI)16501280 (PubMedID)
Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2017-12-14Bibliographically approved
5. An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation: An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation
Open this publication in new window or tab >>An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation: An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation
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2007 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 7, p. 2888-2895Article in journal (Refereed) Published
Abstract [en]

Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experiments, but data analysis is time-consuming and labor-intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administration of reserpine, a classical model drug for inducing Parkinson disease symptoms.

Keywords
LC-MS; quantitation; differential display; neuropeptides; DeCyder MS; label-free quantitation
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-95136 (URN)10.1021/pr060676e (DOI)000247792300049 ()
Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2018-01-13Bibliographically approved
6. Decreased striatal levels of PEP-19 following MPTP lesion in the mouse.
Open this publication in new window or tab >>Decreased striatal levels of PEP-19 following MPTP lesion in the mouse.
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2006 In: J Proteome Res., Vol. 5, no 2, p. 262-269Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-95137 (URN)
Available from: 2006-11-17 Created: 2006-11-17Bibliographically approved

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