Year

2001

Degree Name

Doctor of Philosophy

Department

Department of Chemistry

Abstract

This thesis explores three different applications of electrospray ionisation mass spectrometry (ESI-MS) for protein analysis. An example of the identification of proteins following separation by 2-dimensional electrophoresis is presented with a study of proteins released from mitochondria following low-conductance permeability transition (LC-PT). A number of mitochondrial matrix proteins were identified from gels of the supernatant of mitochondria that had not undergone LC-PT. These findings raise questions regarding the release of proteins from normally functioning mitochondria. A second possibility is that the integrity of some of the mitochondrial membranes in the cell-free system may have been compromised. The identification of peroxisomal proteins in the mitochondrial preparations highlighted a major problem that is encountered with subcellular fractionation of organelles, namely contamination from other cellular components. This study also demonstrated the need for automated procedures to minimise human contamination and obtain reproducible results.

Functional proteomics is aimed at obtaining information on binding partners and protein function. An example of a functional proteomics approach developed here involved the use of affinity and ion exchange chromatography in conjunction with MS to identify proteins that associate with the T cell surface protein, CD4. The identification of a known CD4-binding protein, p56lck, provided evidence that the method was suitable for the isolation of CD4-associating proteins. Identification of other interesting proteins (tubulin, myosin, actin, B-cell receptor-associated protein and two ribosomal proteins) has revealed potential CD4-binding partners for further immunological studies.

An interesting area in which ESI-MS has evoked significant interest has been in the analysis of noncovalent complexes. The gentle nature of ESI results in the ability to transfer complexes that are bound noncovalently into the gas phase under carefully controlled conditions. There is, however, still debate as to what extent the gas phase behaviour of complexes reflects their solution phase properties. Glutathione Stransferase (GST) is an ideal protein for investigating the applicability of ESI-MS to this type of analysis. A study of the complexes formed between GST and various ligands by ESI-MS is also presented. This study showed the formation of GST dimers within proteins of the same enzyme class. It was interesting that glutathione did not reveal the expected binding of two molecules per GST dimer. The preferential binding of two Salkylglutathione molecules to the dimer, however, was clear from these data and the increased binding strength with increasing alkyl chain length was also apparent. Tandem mass spectrometry (MS/MS) was also found to be useful for investigating the specificity of ligand binding and for identifying the ligands bound to the GST dimer. MS/MS also showed that S-alkylglutathione ligands with longer alkyl chains required more energy to be removed from the complex that the S-alkylglutathiones of shorter alkyl chain lengths.

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