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Title: The development of mass spectrometry-based methods for the analysis of peptides and proteins
Author: Atwal, Gushinder Kaur
ISNI:       0000 0001 3432 6330
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2008
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The identification of proteins and peptides has become one of the most common applications in biological mass spectrometry. A primary aim of analysts in protein and peptide analysis is to reduce the complexity of sample mixtures in order to enhance sensitivity and selectivity. The research reported in this thesis describes the development of mass spectrometry-based methods for the separation and analysis of peptide mixtures for rapid, high-throughput proteomic analysis. A novel approach has been developed for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation coupled with ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (DESI/IMS/Q-ToF-MS). Confident protein identification was demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge state and mobility distributions for tryptic peptide mixtures. The mobility-selected singlycharged peptide responses were presented as a pseudo-peptide mass fingerprint (pPMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests are discussed. Direct coupling of capillary immobilised metal affinity chromatography (IMAC) with mass spectrometry allowed on-line separation and detection of peptides in a highthroughput fashion, minimising preparative steps and sample handling, while reducing analysis time compared to alternative off-line strategies. This approach has been demonstrated for the isolation of histidine- and oxidised methionine- containing peptides from peptide mixtures using copper (II) and aluminium (III) activated capillary IMAC columns. The on-line capillary IMAC separations were earried out using mass spectrometry compatible loading and elution buffers and analytical procedures were optimised to prevent metal ion leaching from the IMAC column. The potential of ion mobility/mass spectrometry combined with IMAC has also been examined, bringing an additional selectivity dimension to the analysis, allowing charge-state separation and enhanced MS detection of biomolecules.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available