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- Fridjonsdottir, Elva, et al.
(författare)
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Mass spectrometry imaging reveals brain-region specific changes in metabolism and acetylcholine levels in experimental Parkinson’s disease and L-DOPA-induced dyskinesia
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- There is evidence that cholinergic alterations are linked to various motor and non-motor symptoms of Parkinson’s disease. We therefore used mass spectrometry imaging to investigate regional changes in acetylcholine abundance in the brain of a non-human primate model of Parkinson’s disease (PD) and L-DOPA-induced dyskinesia (LID). We also present an experimental design for performing untargeted analysis using MALDI-MSI with multiple experiments incorporating quality control samples to monitor experimental variability. We observed that MPTP treatment (i) led to reductions in putaminal acetylcholine levels that persisted after L-DOPA treatment and (ii) appeared to induce a shift of choline metabolism from α-glycerophosphocholine towards betaine. LID animals exhibited reduced levels of various metabolites important for brain homeostasis including S-adenosylmethionine, glutathione, adenosine monophosphate, and acylcarnitines. The vasculature marker heme B was upregulated in the putamen of LID animals, suggesting increased blood-flow in the dyskinetic putamen. These results provide new insights into pathological choline-related metabolic changes in PD and LID.
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- Fälth Savitski, Maria, 1979-
(författare)
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Improved Neuropeptide Identification : Bioinformatics and Mass Spectrometry
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bioinformatic methods were developed for improved identification of endogenous peptides using mass spectrometry. As a framework for these methods, a database for endogenous peptides, SwePep, was created. It was designed for storing information about endogenous peptides including tandem mass spectra. SwePep can be used for identification and validation of endogenous peptides by comparing experimentally derived masses of peptides and their fragments with information in the database. To improve automatic peptide identification of neuropeptides, targeted sequence collections that better mimic the peptidomic sample was derived from the SwePep database. Three sequence collections were created: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, and it was observed that three times as many peptides were identified with the targeted SwePep sequence collections. Applying the targeted SwePep sequence collections to identification of previously uncharacterized peptides yielded 27 novel potentially bioactive neuropeptides.Two fragmentations studies were performed using high mass accuracy tandem mass spectra of tryptic peptides. For this purpose, two databases were created: SwedCAD and SwedECD for CID and ECD tandem mass spectra, respectively. In the first study, fragmentation pattern of peptides with missed cleaved sites was studied using SwedCAD. It was observed that peptides with two arginines positioned next to each other have the same ability to immobilize two protons as peptides with two distant arginines. In the second study, SwedECD was used for studying small neutral losses from the reduced species in ECD fragmentation. The neutral losses were characterized with regard to their specificity and sensitivity to function as reporter ions for revealing the presence of specific amino acids in the peptide sequence. The results from these two studies can be used to improve identification of both tryptic and endogenous peptides.In summary, a collection of methods was developed that greatly improved the sensitivity of mass spectrometry peptide identification.
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- Källback, Patrik
(författare)
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Development and Application of Software Tools for Mass Spectrometry Imaging
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mass spectrometry imaging (MSI) has been extensively used to produce qualitative maps of distributions of proteins, peptides, lipids, neurotransmitters, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. Moreover, during the last 10 years, there has been growing demand to quantify target compounds in tissue sections of various organs. This thesis focuses on development and application of a novel instrument- and manufacturer-independent MSI software suite, msIQuant, in the open access format imzML, which has been developed specifically for quantitative analysis of MSI data. The functionality of msIQuant facilitates automatic generation of calibration curves from series of standards that can be used to determine concentrations of specific analytes. In addition, it provides many tools for image visualization, including modules enabling multiple interpolation, low intensity transparency display, and image fusion and sharpening. Moreover, algorithms and advanced data management modules in msIQuant facilitate management of the large datasets generated following rapid recent increases in the mass and spatial resolutions of MSI instruments, by using spectra transposition and data entropy reduction (at four selectable levels: coarse, medium, fine or superfine) before lossless compression of the data. As described in the thesis, implementation of msIQuant has been exemplified in both quantitative (relative or absolute) and qualitative analyses of distributions of neurotransmitters, endogenous substances and pharmaceutical drugs in brain tissue sections. Our laboratory have developed a molecular-specific approach for the simultaneous imaging and quantitation of multiple neurotransmitters, precursors, and metabolites, such as tyrosine, tryptamine, tyramine, phenethylamine, dopamine, 3-methoxytyramine, serotonin, gamma-aminobutyric acid (GABA), and acetylcholine, in histological tissue sections at high spatial resolution by matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) MSI. Chemical derivatization by charge-tagging primary amines of analytes significantly increased the sensitivity, enabling mapping of neurotransmitters that were not previously detectable by MSI. The two MSI approaches have been used to directly measure changes in neurotransmitter levels in specific brain structures in animal disease models, which facilitates understanding of biochemical mechanisms of drug treatments. In summary, msIQuant software has proven potency (particularly in combination with the reported derivatization technique) for both qualitative and quantitative analyses. Further developments will enable its implementation in multiple operating system platforms and use for statistical analysis.
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- Sköld, Karl, 1974-
(författare)
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Neuropeptidomics – Methods and Applications
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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- Svensson, Marcus, 1972-
(författare)
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Neuropeptidomics – Expanding Proteomics Downwards
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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