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The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin (2-20) and peptides as sample quality indicators.
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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2007 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 24, 4445-4456 p.Article 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.

Place, publisher, year, edition, pages
2007. Vol. 7, no 24, 4445-4456 p.
Keyword [en]
Phosphorylation, Protein, Peptides, Proteomics, Encephalon
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-95134DOI: 10.1002/pmic.200700142ISI: 000252237000005PubMedID: 18072205OAI: oai:DiVA.org:uu-95134DiVA: diva2:169226
Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Neuropeptidomics – Methods and Applications
Open this publication in new window or tab >>Neuropeptidomics – Methods and Applications
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. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 42
Keyword
Pharmaceutical pharmacology, mass spectrometry, proteomics, peptidomics, neuropeptide, bioinformatics, Farmaceutisk farmakologi
Identifiers
urn:nbn:se:uu:diva-7276 (URN)91-554-6717-2 (ISBN)
Public defence
2006-12-08, B42, BMC, Husargatan 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2006-11-17 Created: 2006-11-17Bibliographically approved
2. Neuropeptidomics – Expanding Proteomics Downwards
Open this publication in new window or tab >>Neuropeptidomics – Expanding Proteomics Downwards
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological function is mainly carried out by a dynamic population of proteins which may be used as markers for disease diagnosis, prognosis, and as a guide for effective treatment. In analogy to genomics, the study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel based proteomics is methodologically restricted from the low mass region which includes important endogenous peptides. Furthermore, the study of endogenous peptides, peptidomics, is compromised by protein fragments produced post mortem during conventional sample handling.

In this thesis nanoflow liquid chromatography and mass spectrometry have been used together with improved methods for sample preparation to semi-quantitatively monitor peptides in brain tissue. The proteolysis of proteins and rise of fragments in the low mass region was studied in a time-course study up to ten minutes, where a potential marker for sample quality was found. When rapidly denatured brain tissue was analyzed, the methods enabled detection of hundreds of peptides and identifications of several endogenous peptides not previously described in the literature. The identification process of endogenous peptides has been improved by creating small targeted sequence collections from existing databases.

In applications of the MPTP model for Parkinson’s disease the protein and peptide expressions were compared to controls. Several proteins were significantly changed belonging to groups of mitochondrial, cytoskeletal, and vesicle associated proteins. In the peptidomic study, the levels of the small protein PEP-19 was found to be significantly decreased in the striatum of MPTP administered animals. Using imaging mass spectrometry the spatial distribution of PEP-19 was found to be predominant in the striatum and the levels were concordantly decreased in the parkinsonian tissue as verified by immunoblotting.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 46 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 48
Keyword
Pharmaceutical pharmacology, proteomics, peptidomics, mass spectrometry, neuropeptide, chromatography, Farmaceutisk farmakologi
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-7465 (URN)978-91-554-6791-1 (ISBN)
Public defence
2007-03-02, B:42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2007-02-09 Created: 2007-02-09 Last updated: 2010-01-15Bibliographically approved

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