Two unique features of FT-ICR mass spectrometry are high mass resolution and mass accuracy. These features have been exploited in a novel approach to the study of metal exchange reactions in metallo-proteins using stable isotope labelling. The system chosen, Zn<sub>4</sub>-SmtA, is a cyanobacterial metallothionein (MT) Deconvoluted ESI-FT-ICR spectra for Zn<sub>4</sub>-FT-ICR spectra for Zn<sub>4</sub>-Smt containing Zn isotopes in natural abundance as well as enriched with 93% <sup>67</sup>Zn show that the Zn<sub>4</sub> cluster of SmtA, in contrast to the structurally-analogous cluster of mammalian MT, contains a kinetically-inert Zn site, a feature which can be related to its secondary and tertiary structure, and which is of potential importance to its biological function. The main body of this work is a systematic study of the dissociation, via ECD, of doubly protonated peptides, such as luteinising hormone releasing hormone (LHRH), bradykinin and bombesin. The most common fragments reported in the literature occur from cleavage of the N-C<sub>12</sub> backbone bond, yielding c’ (N-terminal) and z’ (C-terminal) fragments. One striking observation in the spectra of the peptides studied is the presence of prominent radical c fragments (labelled herein c’) and even-electron z fragments (labelled z’). A study of the correlation between the relative abundance of each of the fragment ions as a function of the amino acid sequence for each of the peptides is shown. These experimental studies have been complemented molecular mechanics modelling, using the Amber force field, to correlate with putative gas-phase structures for these doubly protonated peptides. Here, a hypothesis is put forward that the relative ECD fragmentation pattern is related to the gas-phase structure of the peptide ion <i>prior to</i> electron capture, and in particular that hydrogen bonding of the protonated site to the backbone enhances backbone cleavage at that site (referred to as ‘ECD/structure correlation’). The relative ECD fragmentation pattern for bombesin and four LHRH variants are compared to their modelled gas-phase structures. A detailed study of the LHRH variants with sequence pEHWSYGLRPG-OH {1} and pEHWSYGLRPG-NH<sub>2</sub> {2}, and bombesin {3}, showed good agreement between the relative ECD fragment abundances and predicted Amber structures for {1} and {3}, but not for {2}. This may imply that the postulated model is not a full description of the ECD process.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660656 |
| Date | January 2003 |
| Creators | Polfer, Nicolas Camille |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11267 |
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