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Title: LC-MS analytical strategies for chemical speciation in complex biological samples
Author: Siemieniako, Lidia K.
ISNI:       0000 0004 2724 8196
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2010
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This work investigates a variety of hyphenated strategies for studying metal protein binding in complex biological samples. Special attention was given to analysis of lanthanum and zinc. Chapter One describes the various roles played by metal ions in biological systems. The importance of total metal concentration measurements in biofluids as well as metal speciation studies, are outlined. Methods for trace element determination as well as biomolecule characterisation are covered. Separation techniques used for biological samples are described with special attention to electrophoresis and liquid chromatography (LC). Strategies for elemental speciation in biological samples are outlined with particular consideration to techniques currently employed. Chapter Two describes development of a high throughput methodology for ultratrace analysis of La in biofluids by liquid nebulisation ICP-MS, which was found to be suitable for the determination of total La concentration in biofluids (LOD 8.6 ng/L). Good analytical performance for the analysis of aqueous La standards was also achieved by laser ablation of dried droplets. Chapter 3 describes in vitro studies on La speciation in human serum. Two dimensional polyacrylamide gel electrophoresis (20 PAGE) followed by an electroelution and flow injection ICP-MS analysis of harvested fractions encountered problems associated with La contamination as well as poor sample recovery. This work also investigates direct interrogation of gels by laser ablation-ICP-MS which suggested extensive protein binding of La by serum proteins. The second part of this chapter investigates the coupling of anion exchange chromatography to ICP-MS which suggested that La is bound to one of transferrin isoforms. Chapter Four describes the development of two dimensional liquid chromatography for separation of complex biological samples (bacterial cell lysates). The separation method was assessed by comparison of control and ZInC stressed E. coli cell lysates. Chapter five describes the use of two dimensional LC method linked to MS for characterisation of proteins in bacterial cell lysates. Different MS platforms were assessed for analysis of intact species ('top down' proteomics) and tryptic digests (,bottom up' proteomics). Separation and sample requirements were compared for ESI and MALDI MS. LC fractions were collected for off-line MALDI analysis, or the flow was coupled directly to ESI-MS for on-line analysis. Intact species and tryptic digests were analysed. Chapter Six considers LC-ICP-MS strategies for characterisation of zincbinding proteins in bacterial cell lysates. Effects of organic solvents in ICP-MS for analysis of zinc are evaluated and off-line and on-line ICP-MS strategies were exploited. However, the developed 2D-LC coupled to ICP-MS experiments have, as yet, not permitted quantitation of the zinc containing proteins in the E. coli cell lysates.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available