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Title: Chromatographic, capillary electrophoretic and mass spectrometric studies of proteins and smaller biomolecules
Author: Hunter, A. P.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1994
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Chapter One introduces chromatography and mass spectrometry. The separation mechanisms of liquid chromatographic techniques are reviewed. The fundamentals of mass spectrometry, mass analysers, scanning modes and ionisation methods, and means of interfacing liquid chromatography and mass spectrometry are discussed, with particular reference to biomolecular analysis. Chapter Two provides a review of the development of capillary electrophoresis and capillary electrophoresis/mass spectrometry. The construction of a co-axial sheath flow interface for capillary electrophoresis/electrospray-mass spectrometry and liquid chromatography/electrospray mass spectrometry is detailed. The efficiency and efficacy of each system for biomolecular analysis is discussed. Chapter Three demonstrates the capillary electrophoretic separation of nucleosides and nucleotides using conventional and micellar buffer systems. Capillary electrophoretic/mass spectrometric analysis of nucleoside and isomeric nucleotide mixtures is demonstrated, and partial characterisation of the nucleotide positional isomers effected by electrospray tandem mass spectrometry. Chapter Four documents an investigation into the characterisation of phosphorylation sites in bovine α-casein. Electrospray and laser desorption mass spectrometry assess the molecular weight and phosphate quotient of the intact protein. Comparative peptide mapping of phosphorylated and dephosphorylated α-casein proteolytic digest by capillary electrophoresis/mass spectrometry and liquid chromatography/mass spectrometry is shown to yield information on siting of phosphate groups, and selective identification of phosphopeptides is demonstrated using liquid chromatography/collision induced dissociation (in the transport region of the electrospray source)/mass spectrometric techniques. The utility of Edman sequencing and tandem MS of phosphopeptides for elucidation of the phosphate position is established. Chapter Five details the identification of glycosylation sites and heterogeneity in two bovine glycoproteins: fetuin and α-acid glycoprotein. Glycosylation siting and heterogeneity within the intact and digested glycoproteins are evaluated using capillary electrophoresis, and as in Chapter Four, the important role of mass spectrometry for selective identification of glycopeptides is again emphasised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available