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Title: The mass spectrometry of selected proteins and peptides
Author: Millar, Alan L.
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1998
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The multi-subunit alkene monooxygenase was studied by means of electrospray ionisation mass spectrometry (ESI-MS). The measured relative molecular mass (RMM) values for the epoxygenase and reductase were in good agreement the predicted RMM values calculated by means of gene sequencing. Cleavage sites were identified by means ofESI-MS at the N- and C- terminus of the coupling protein. Cleavage at the Cterminus inactivated the protein but was shown to be prevented by the addition of a protease inhibitor, benzamidine. The ceroid lipofuscinosis protein (CLP) was found to contain a +42.2 Da modification. Low energy tandem mass spectrometry confirmed 80% of the primary sequence of the CLP, indicating that the discrepancy in RMM is located on amino acid residues 30-46. Bovine heart mitochondrial subunit c was extracted as a control and was also found to contain the +42.2 Da modification. A study of the interaction ofCLP with its diagnostic inhibitor DCCD was performed. This indicated that the primary product of the reaction was an acetyl-CLP complex and not the expected CLP-DCCD complex. The p-subunit of the soluble methane monooxygenase (sMMO) was investigated by means of ESI-MS to locate and identify a +126.3 Da discrepancy in RMM. A tryptic digestion of the protein indicated the discrepancy to be located in a small digest peptide consisting of amino acid residues 141-146. Low energy MSIMS was used to correct the primary sequence of this peptide. The revised sequence was supported by the close similarity between the DNA nucleotide sequence for the originally suggested amino acid residue sequence and the revised sequence. The binding between a 17 amino acid micro-antibody (Micro-Ab) and two peptides which represent the V3 region of the gp 120 external glycoprotein of the HIV-l envelope protein were studied by means ofESI-MS. It was found that the interaction between the Micro-Ab and the two V3 peptides could be maintained and thus observed directly by ESI-MS. A rapid method of epitope identification was devised whereby the antigen was pre-digested before incubation with the Micro-Ab. The data identified a similar series of amino acid residues in both V3 peptides as the epitope with which the Micro-Ab interacts.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council (EPSRC) ; Micromass UK ; British Mass Spectrometry Society (BMSS) ; American Study and Student Exchange Committee (ASSEC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QR Microbiology Biochemistry