Developing tissue proteomics: Differential in gel electrophoresis in biomarker discovery and proteomic degradation

Q Science (General)

Lang, Alastair Michael ; Pitt, Andrew R

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: Proteomics, Differential in-gel electrophoresis, degradation, tissue, biomaker;   

Others  :  http://theses.gla.ac.uk/4642/1/2013Langphd.pdf

来源: University of Glasgow

【 摘 要 】

The field of proteomics and functional genomics has developed steadily since the completion of the human genome project.The wealth of genomic information and the pace at which it was compiled was astounding.Proteomics, despite considerable effort, on the other hand has not seen quite the same pace of development.The progress being considerably hindered by the lack of an amplification process and the relative complexity of the proteome in comparison to the genome.These intrinsic difficulties have led to the sensitivity of proteomic techniques being pushed closer to physical limits.There is therefore a further need to re-evaluated techniques such as sample preparation and integrity, analytical methods and collaborative strategies to maximise the effectiveness and quality of data collected.The importance of tissue in scientific and clinical research is unequivocal.However, tissue is difficult to collect, store and work with due to issues with proteomic degradation and storage.Good lab practices can minimise the effect of degradation but degradation of proteins can be rapid.Strategies to minimise degradation include freezing, formalin fixing and microwave treatment which all have their relative advantages and disadvantages.The importance of sample preparation as being the top of the workflow is often acknowledged but improvements are not well described in the literature.The main aim of this thesis is to present investigative studies into the mitigation of some of the limitations in tissue sample degradation, analytical approaches in differential in gel electrophoresis and accessing DiGE spot and tissue profile data. Presented is the evaluation of the effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and to aid in the cessation of proteomic degradation.A multifaceted analytical approach of differential in Gel electrophoresis spot data is assessed, giving proteomic profiles of mouse brain tissue.Preliminary data is presented showing that the process of heat-treatment has had a predominantly beneficial effect on mouse brain tissue, with a higher percentage of spots stabilised in heat-treated samples compared to snap-frozen samples.However, stabilisation did occur in snap-frozen samples for different protein spot so the appropriateness of using heat-treatment is as yet not fully determined and requires further analysis.In addition, the variation in tissue profiles of WKY, SP.WKYGla.2a and SHRSP rat model for hypertension is investigated with the future prospect of providing that vital connection between genomic and proteomic data and link phenotype and genotype preliminary investigated.A number of putative markers were identified and quantified using DiGE analysis.In order for these markers to be accepted as biomarkers, more downstream validation is required, however this study provides a good spring board as a proof of concept in using DiGE as an global putative biomarker discovery platform.