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Analysis of natural products by high resolution Fourier transform-ion cyclotron resonance mass spectrometry
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Li, Meng (2020) Analysis of natural products by high resolution Fourier transform-ion cyclotron resonance mass spectrometry. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3517409~S15
Abstract
The work presented herein focusses on the study of natural products (from proteins to small molecules) and applies advanced techniques in ultrahigh resolution Fourier transform - ion cyclotron resonance mass spectrometry (FT-ICR MS) to the study the proteomics of venoms as well small drug molecules derived from botanicals.
The venoms obtained from scorpions are well known to contain a complex mixture of compounds that would include salts, organic molecules, and a wide range of proteins. The study of the protein components of venoms have been very popular due to their clinical relevance and wide and biodiversity, which could become important pharmaceutical leads in the development of new drugs.
In addition, venom proteins have demonstrated significant therapeutic effects with high specificity and relatively low toxicity. To better understand the components present in the venom, a couple of different analytical techniques was employed in additional to standard proteomics methodologies to systematically characterise these proteins.
The proteins and peptides identified from the scorpion species, Mesobuthus martensii, from previous only represent a small fraction of the components in the scorpion venom, therefore, it is important to attempt to obtain as much information from the the proteins present in the scorpion venom as possible.
In addition, theses venom proteins are heavily modified and generally contains 3-4 disulphide bond for a 4 kDa protein. This coupled with a relatively small database (113 entries on Uniprot), makes tandem MS very challenging, and requires de novo sequencing of some of the proteins and peptides.
As new methodologies develop for the analysis of proteomics, optimisation of the method to apply to the specific type of samples is necessary. The use of two dimensional mass spectrometry (2DMS) and its application for the study of venoms is explored. 2DMS to crude scorpion venom digest (bottom-up) as well as intact crude scorpion venom proteins (top-down) was studied and the results was compared to those obtained from standard proteomics methods. The ability to use of a wide range of sample preparation methods prior to 2DMS acquisition was explored and demonstrated. The ability to observe more species in 2D as compared to direct infusion 1D has show unique capability of 2DMS in the application of venom studies, and allow for future application to other types of complex mixtures.
For complex samples such as venom studies, the high sequence variance, heavy post-translation modifications and the limited number of database entries, has resulted in the slow and difficult process when seeking possible pharmaceutical targets
The application of multivariate analysis to establish the correlation between the abundance of the proteins present to the bioactivity of the samples is therefore a more efficient method to obtain potent biomolecules, for different treatments, such as anticoagulation. With our methodology, novel proteins with unique post-translation modifications were identified to have strong correlations with anticoagulant bioactivity, indicating potential pharmaceutical targets.
Previous studies focusing on the screening for bioactive species in venoms have been very time consuming, requiring multiple fractionations, coupling to multiple in vitro assays, and optimising LC runs with multiple columns. By using MS data coupled to bioactivity assay and chemometrics, has allowed for the identification of novel bioactive species. The use of FT-ICR MS provided specificity of the species responsible for the bioactivity, i.e. the inhibition of FXa. With our methodology only one crude fractionation was necessary, and is coupled with only 1 in-vitro assay. With chemometrics, reliable results can be obtained in terms out of revealing bioactive species.
Small molecules derived from natural products also plays an important role in drug design and development. The use of MS and MS/MS characterisation of these novel therapeutic compounds is relevant to both the structural and chemical characterisation of these natural product derived compounds products, but also to provide fragmentation pattern information which can be further applied to other compounds of interest in future studies, especially compounds with similar functional groups.
In addition, it can be used for their identification in complex mixtures and tracking within in vivo systems. By characterising these fragments and products using high accuracy and varied techniques allows future application to move towards higher throughput analysis.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Library of Congress Subject Headings (LCSH): | Proteins -- Analysis, Peptides -- Analysis, Ion cyclotron resonance spectrometry, Fourier transform spectroscopy, Scorpions -- Venom | ||||
Official Date: | January 2020 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | O'Connor, Peter B. | ||||
Format of File: | |||||
Extent: | xxii, 263 leaves : illustrations (some colour) | ||||
Language: | eng |
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