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dc.contributor.advisorBowie, John Hamiltonen
dc.contributor.advisorHoffmann, Peteren
dc.contributor.authorAndreazza, Hayley Joen
dc.description.abstractThe identification and characterisation of post-translational modifications (PTMs) of proteins and peptides is vital to understanding their functional properties and complex biological problems. The work presented in Chapters 2-4 of this thesis describes the development and application of a joint negative ion electrospray ionisation tandem mass spectrometry (ESI-MS/MS) and theoretical study into the identification and characterisation of several PTMs. Chapter 2 deals with the characteristic negative ion fragmentations of deprotonated peptides containing mono- and di-phosphorylated systems. The characteristic fragmentations of monophosphorylated peptides containing pSer and pThr are the loss of H₃PO₄ from the (M-H)⁻ anions and the formation of H₂PO₄⁻ (previously identified by Lehmann, et al.). These characteristic cleavages were found to be more energetically favourable than the negative ion backbone cleavages of peptides described previously. The characteristic loss of HPO₃ from pTyr-containing peptides was found to be comparable with those already reported for negative ion backbone cleavages. The (M-H)⁻ anions from selected diphosphopeptides show characteristic peaks corresponding to m/z 177 (H₃P₂O₇⁻), 159 (HP₂O₆⁻) and sometimes [(M-H)⁻-H₄P₂O₇]⁻. The characteristic fragmentations of a pTyr group in the negative ion electrospray tandem mass spectrum of the (M-H)⁻ parent anion of a peptide or protein involve the formation of PO₃⁻ (m/z 79) and the corresponding [(M-H)⁻-HPO₃]⁻ species. In some tetrapeptides where pTyr is the third residue, these characteristic anion fragmentations are accompanied by peaks corresponding to H₂PO₄⁻ and [(M-H)⁻-H₃PO₄]⁻ (these are fragmentations normally indicating the presence of pSer or pThr). These fragment ions are formed by rearrangement processes which involve initial nucleophilic attack of a C-terminal -CO₂⁻ [or C (=NH)O⁻] group at the phosphorus of the Tyr side chain [an Sɴ2(P) reaction]. Chapter 3 describes how the negative ion ESI-MS of the peptides produced by tryptic and chymotrypsin digests of bovine insulin, and from the tryptic digest of lysozyme identify at least 80% of the sequences of these proteins as well as the positions of disulfide moieties. Chapter 4 reports on the experimental and theoretical investigation into the negative ion fragmentations of Asp and isoAsp. It was found that it is not possible to differentiate between Asp and isoAsp containing peptides (used in this study) using negative ion ESI-MS because the isoAsp residue cleaves to give the same fragment anions as those formed by δ and γ backbone cleavage of Asp. No diagnostic cleavage cations were observed in the electrospray mass spectra of the MH+ of the Asp and isoAsp containing peptides (used in this study) to allow differentiation between these two amino acid residues. Chapter 5 describes the mass spectrometric component of a study showing that some selected His-containing anuran peptides, namely caerin 1.8, caerin 1.2, Ala₁₅ maculatin 1.1, fallaxidin 4.1, riparin 5.1 and signiferin 2.1, all form MMet²⁺ and (M+Met²⁺-2H⁺)²⁺ cluster ions (where Met is Cu, Mg and Zn) following ESI. Peaks due to Cu (II) complexes are always the most abundant relative to other metal complexes. Information concerning metal²⁺ connectivity in a complex was obtained using b and y fragmentation data from CID ESI-MS/MS.en
dc.subjectmass spectrometry; post-translational modifications; peptides; electrospray ionisationen
dc.titleCharacteristic negative ion fragmentations of deprotonated peptides containing post-translational modifications.en
dc.contributor.schoolSchool of Chemistry and Physicsen
dc.provenanceThis 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:
dc.description.dissertationThesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2014en
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