Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.569795
Title: N-terminal plasma profiling : high efficiency plasma proteomics
Author: Davidson, Gemma Rose
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2011
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Abstract:
Blood is a highly accessible body fluid that is rich in biological information; it is widely accepted that this is the most popular diagnostic sample available within a clinical environment. The measurement of cellular and non-cellular blood components, including blood cells, electrolytes, metabolites, lipids and proteins can aid clinicians in establishing an individuals' health status and can also function to diagnose and monitor the progression of disease. An alteration in an individuals' physiological state, such as the presence/absence of disease, will often lead to an alteration in protein expression. The accurate identification and measurement of proteins found in the non-cellular component of blood, known as plasma, could provide key information relating to a multitude of biological processes. Blood plasma is considered to contain the most comprehensive proteome of the body, as it is in contact with every cell, tissue and organ. Enthused by the clinical potential of this biological fluid there has been a drive within the proteomics community to develop robust proteomic platforms for the analysis of the blood plasma proteome. Coupled with powerful technologies, such as chromatography and mass spectrometry, there has been some success. Initiatives, such as the human plasma proteome project (PPP) have strived to standardised and collate data across laboratories with the objective of the global analysis and quantification of the plasma proteins. Unfortunately, as yet the plasma proteome remains to a large degree undiscovered, or more suitably 'hidden'. Blood plasma contains many proteins from all parts of the body. Tensioned against these desirable features are two disadvantages that have thus far hindered most plasma proteomic studies: high complexity and large dynamic range. The quantitative dynamic range of the human plasma proteome spans over 10 orders of magnitude with a small number of high-abundance proteins dominating approximately 99% total protein concentration, with albumin alone constituting approximately 50% total plasma protein. The dominance of the plasma proteome by such high-abundance proteins has inhibited the ability of most proteomic studies to access those proteins considered low-abundance. This is especially problematic when one considers that those proteins that constitute approximately 1 % total plasma protein, such as tissue leakage proteins, are considered to be extremely important in protein biomarker discovery. The analytical challenges imposed by blood plasma have thus far hampered efforts to delve deep into the proteome; presently no analytical technique has been presented that can fully exploit the full potential of plasma for medicine and therapeutics. A reduction in analyte complexity and improvements in analytical instrumentation are the most common approaches to combat the complexity and dynamic range problems associated with the study of the plasma proteome. Although the selective removal of high-abundance proteins via immunodepletion is an attractive approach to address the dynamic range issue, many of the high-abundance plasma proteins, such as albumin, have been shown to associate with other proteins. Thus, selective depletion will lead to the non-specific removal of other proteins. Other strategies aim to selectively target specific amino acid residues for enrichment, however utilising such an approach might lead to the over or under-representation of certain proteins. A positional proteomics approach targets one of two locations common to all proteins; the N- terminus or the C-terminus. This investigation focuses on the adoption of a positional proteomics strategy to selectively enrich the N-terminal peptides of the human plasma proteins. A reduction in sample complexity is observed when compared to a traditional 'shot-gun' proteomic approach. The absolute measurement of proteins is particularly important for protein biomarker validation/verification and for the development of our own understanding of biological processes. The development ofa QconCAT protein that is a concatomer ofN-terminal peptides, each a surrogate for its parent plasma protein, has the potential to provide absolute measurement of N-terminal peptides in a multiplexed manner. Utilising highly sensitive and selective mass spectrometric techniques such as selected reaction monitoring (SRM) in combination with the N-terminal enrichment strategy and QconCAT technology there is potential to selectively target and quantify N- terminal peptides in a high-throughput manner. Utilising N-terminal enrichment as a tool for human plasma proteome analysis gives an insight into the magnitude of enzymatic processes that specifically target the N-terminal portion of a protein. Such exo- and endo- proteolytic activity could prove significant for biomarker research. Although dynamic range is still a major problem, the N-terminal enrichment strategy demonstrates its utility as a tool for profiling the human plasma N-terminome. The global analysis of the plasma proteome will most likely require a combination of proteomic approaches that will specifically target and enrich sub-sections of this unique proteome.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.569795  DOI: Not available
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