Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663616
Title: FT-ICR mass spectrometry of metalloproteins
Author: Weidt, Stefan K.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Abstract:
Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) has been used to study a number of metalloproteins. The combination of high-resolution and mass-accuracy, along with fragmentation techniques such as CID, IRMPD and ECD, is shown to be a powerful method to probe metalloproteins and metallodrug interactions. The inherent high resolving power and mass measurement accuracy of the FT-ICR MS also enables the oxidation state of the metal present in a metalloprotein to be identified from the isotopically resolved spectra. Carbonic anhydrase II (29 kDa) is an enzyme that catalyses the reversible hydration of carbon dioxide and contains one Zn2+ ion. To investigate non-covalent protein-drug interactions, the inhibitor acetazolamide was added to a sample of carbonic anhydrase II. Nozzle-skimmer CID, IRMPD and titration methods were used to probe the strength of interaction of the carbonic anhydrase-acetazolamide complex. It was found that lower charge-states bind more strongly than higher charge-states suggesting that the protein unfolds when more protons are bound. FT-ICR MS was used to investigate the nature of adducts between the platinum anti-cancer drug cisplatin, and SOD. The structure showed a cisplatin adduct at His19 of beSOD, which had retained the chloride ligands; these are usually lost in aqueous conditions. After incubation with cisplatin, the mono-platinated species was the most prominent adduct observed. The use of 15N-labelled cisplatin led to unambiguous assignment of the two ammonia ligands being retained after binding to beSOD, supporting established cisplatin solution behaviour. Mass spectra of heSOD were more complex than those for beSOD due to phosphate buffer adducts and the loss or substitution of the chloride ligands. ECD fragmentation spectra of the isolated mono-platinum adduct of beSOD were used in order to localise the site of modification.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.663616  DOI: Not available
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