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Title: Enhancement of the proteomics workflow and application to stem cell characterisation
Author: Techanukul, Tanasit
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Stem cell research promises to transform current medical practices by providing alternative solutions for degenerative diseases, bodily injuries, organ failures, and cancers. One area that has been most extensively researched over the past decades is the use of genomics and transcriptomics to unravel the underlying molecular mechanisms of stem cell biology. However there are a number of reasons why information at the DNA and mRNA levels alone does not provide a complete profile of cellular activities. Proteins are ultimately the main functional macromolecules in living organisms and therefore study at the protein level is of importance in providing a more complete understanding of cellular behaviour. Recent developments in proteomics technology provide the tools to analyse functional protein profiles and determine key molecular factors in stem cell maintenance and differentiation processes. To date the current state-of-art proteomics approaches provide powerful tools for investigating the molecular basis of stem cell behaviour for both fundamental and applied research. The improvement in proteomic technologies is a key component in understanding stem cell biology, which is paving the way for the generation of unlimited cells of specific phenotypes for cell-based therapy and incorporation into engineered tissue constructs. This thesis will focus on the improvement of gel-based proteomics technology and the applications of proteomics in human embryonic stem cell research. A general workflow for gel-based proteomics is as follows: [Workflow structure diagram appears here. To view, please open pdf attachment] Chapter 1 includes an introduction in general to proteomics and stem cell biology. Chapter 2, 3 and 4 illustrate experimental results in the improvement of gel-based proteomics workflow. These improvements are in the areas of protein sample quality screening, economical alternatives for pre-stained fluorescent labelling in electrophoretic separations, and suitable image analysis software for fluorescent gel-based electrophoresis, respectively. In chapter 2, a new approach to screen and quickly analyse the quality of samples before running 2-DE was developed to save valuable time and materials. Multi-pixel detection combined with capillary electrophoresis was used as a screening tool for analysing the quality of samples prior to 2-DE. The results showed high resolution and reproducibility, accurate molecular weight assignment, relative quantitation, and absolute quantification of known proteins. This work is an important step towards the standardisation of gel-based proteomics, providing a rapid and visual evaluation method for the quality assessment of 2-DE samples. In chapter 3, an alternative set of cyanine dyes, which have similar chemical properties and a price much lower than the DIGE CyDyes, was investigated for comparative quantitation capability. Through multiple sample analyses these dyes were demonstrated to produce practically identical results to DIGE CyDyes, which allowed sample multiplexing and accurate quantitation for differential proteome expression analysis. This alternative set of cyanine dyes provides a useful approach for staff training, optimisation of experimental methods, and for preliminary results. In chapter 4, three commonly available DIGE enabled software packages were compared of their performance in DIGE experiments. The results demonstrated that all three software packages performed in a generally satisfactory manner, each with strengths and weaknesses. However, based on matching accuracy, Progenesis SameSpots software outperformed the other two software packages, possibly benefiting from its unique algorithm by outlining identical spot across all the gels. Chapter 5 presents comparative proteome profiles of human embryonic stem cells and human induced pluripotent stem cells using gel-based proteomics that provide insight into stem cell biology. The proteomic results showed that 24 protein spots from the gel between two cell populations differentially expressed more than two-fold change, being relatively 22 down-regulated and 2 up-regulated in iPSC population. Of those 24 spots, two identified proteins, being ATP dependent DNA helicase II protein (Ku80 protein) and heat shock 70kDa protein 9 protein (mortalin protein), were selected for further validation by q-PCR and western blotting. The final chapter gives a summary and suggests future direction of the work. Taken together, the work in this thesis has produced four experimental publications (three published and one intended for publication in Journal of Cellular Biochemistry) in the enhancement of gel-based proteomics workflow and the application of proteomics in stem cell biology to decipher the relationship between the molecular phenotype of native and induced pluripotent stem cells.
Supervisor: Cass, Tony Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral