Structural and mechanistic studies of post-translationally modified peptides and proteins.
| dc.contributor.advisor | Bowie, John Hamilton | en |
| dc.contributor.advisor | Pukala, Tara Louise | en |
| dc.contributor.author | Tran, Thi Thanh Nha | en |
| dc.contributor.school | School of Chemistry and Physics | en |
| dc.date.issued | 2014 | en |
| dc.description.abstract | In mass spectrometry (MS), negative ions can be formed by many ion sources, and although sometimes less predominant than their cationic counterparts, they can be observed and studied to provide complementary molecular, ionic structure and mechanistic information. The research presented in this thesis investigates the production and use of negative ions for the structural determination of underivatised peptides and proteins and some post-translationally modified peptides and proteins. An additional application of this research is to determine the structure and membrane interaction of some peptides isolated from Australian amphibians. Phosphorylated Tyr (pTyr) containing peptides undergo SNi [N subscript] cyclisation of the C-terminal carboxylate anion at the P of the pTyr to effect transfer of PO₃H₂ to the C-terminal position. (A similar phosphate rearrangement from pTyr to side-chain carboxylate sites or to the side chains of Ser/Thr also occurs). Following proton transfer, several rearrangements initiated by this phosphate anion can occur, including a specific cyclisation to, and cleavage of, the peptide backbone at the central C of the penultimate amino acid residue. When a peptide contains two/three phosphate side chains, phosphate groups undergo phosphate/phosphate cyclisation to form characteristic di-/tri-phosphate anions. The mechanisms of all fragmentation processes are suggested with the assistance of ab initio theoretical calculations. The major negative-ion fragmentation of Tyr sulfate containing peptides is [(M-H) - SO₃]⁻ and this process normally yields the base peak of the spectrum. Rearrangement reactions involving the formation of HOSO₃⁻ and [(M-H) - H₂SO₄]⁻ yield minor peaks with relative abundances ≤ 10% and ≤ 2% respectively. A Ser sulfate containing peptide, in contrast, shows pronounced peaks due to cleavage product anions [(M-H) - SO₃]⁻ and HOSO₃⁻. Theoretical calculations at the CAM-B3LYP/6-311++g(d,p) level of theory suggest that rearrangement of a Ser sulfate to give C-terminal CO2SO3H is energetically unfavourable in comparison with fragmentation of the intact Ser sulfate to yield [(M-H) - SO₃]⁻ and HOSO₃⁻. [(M-H) - H₂SO₄]⁻ anions are not observed in the spectra of peptides containing Ser sulfate, presumably because HOSO₃⁻ is a relatively weak gas-phase base (∆Gacid = 1265 kJ mol⁻¹). The peaks corresponding to anions formed following cyclisation of the sulfate groups are not detected in the spectra of energised (M-H)⁻ ions of Ser disulfate containing peptides. Proteolytic digest/negative ion nanospray MS was used to determine the five disulfide units and much of the amino acid sequence of ricin, addressing both ricin detection and structural confirmation. Negative ion MS is found to be more effective than positive ion MS in identification and sequencing disulfide bridged peptides. While positive ion MS only provides partial sequences of disulfide containing peptides and often does not specify the positions of disulfide resides, negative ion MS gives clear evidence for the presence and positions of disulfide linkages via characteristic fragmentations. The skin peptide profiles of the red tree frog Litoria rubella (L. rubella) from three locations, namely Flinders Ranges, a region of south-western Queensland and Longreach (Queensland), have been investigated in an eight-month survey. Nine peptides were identified primarily using MS. While the secretion from the L. rubella frogs from Flinders Ranges consists of only the major peptide, tryptophyllin L1.2; the L. rubella frogs from the south-western Queensland and Longreach (Queensland) produce a number of small tryptophyllin peptides and two rubellidins (caeridin type). The primary structures of the major peptide tryptophyllin L1.2 and the two rubellidins (caeridin type) 4.1 and 4.2 were determined previously. The noticeable findings were the discovery of three tryptophyllin metabolite containing peptides including tryptophyllin L1.6, 1.7 and 1.8. The peptide profiles of these frog populations added more information about the evolutionary divergence of this genus. Schwyzer and Zerbe have proposed that certain neuropeptides can transfer from extracellular fluid to attach to a cell membrane prior to moving from that membrane to the adjacent active site of a transmembrane receptor. There are differences in the detailed mechanisms proposed but the key feature is the initial addition of the neuropeptide to the membrane. The Quartz Crystal Microbalance technique with Dissipation (QCM-D) was used to see whether certain amphibian neuropeptides are able to add to a mammalian model bilayer without destroying that membrane. It appears that the peptides may have different modes of interaction with the membrane depending upon overall charges, the charge densities, the secondary structures and the free energies of transferring (to water-membrane interface and to membrane interior), and that the membrane binding may take part but not play a requisite role in a receptor-binding process. | en |
| dc.description.dissertation | Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2014 | en |
| dc.identifier.uri | http://hdl.handle.net/2440/88694 | |
| dc.provenance | This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals | en |
| dc.provenance | Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text. | en |
| dc.subject | negative ion mass spectrometry; phosphorylation sulfation | en |
| dc.title | Structural and mechanistic studies of post-translationally modified peptides and proteins. | en |
| dc.type | Thesis | en |
Files
Original bundle
1 - 3 of 3
No Thumbnail Available
- Name:
- Restricted
- Size:
- 13.71 MB
- Format:
- Adobe Portable Document Format
- Description:
- Library staff access only