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Generation and characterization of cationic and anionic radical peptidesLam, Ngor-wai., 林我威. January 2006 (has links)
published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy
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Gas-phase formation, isomerization and dissociation of peptide radicalcations: energetics, dynamics, and mechanismsSong, Tao, 宋涛 January 2012 (has links)
Understanding the dissociation of odd-electron peptide radical cations is of great importance for the analytical applications of biological mass spectrometry because their diverse array of fragmentation pathways provides structural information to supplement that from even-electron protonated peptides—allowing peptide sequencing and, ultimately, protein identification. Nevertheless, the mechanisms of peptide radical formation and dissociation remain largely unexplored.
In the studies reported in this Thesis, peptide radical cations (M?+) were generated through one-electron transfer (ET) in collision-induced dissociation (CID) of [CuII(L)M]?2+ (L = auxiliary ligand; M = peptide) complexes. Competitive dissociative pathways were circumvented experimentally through judicious selection of the macrocyclic auxiliary ligand, allowing the formation of a broad range of M?+ species. Chapter 3.1 examines the competition between proton transfer (PT) and ET within [CuII(L)His]?2+ complexes with L = dien (an open-chain ligand), or L = 9-aneN3 (the macrocyclic analogue of dien). Density functional theory (DFT) calculations revealed that macrocyclic ligand (9-aneN3) facilitates M?+ formation by maintaining similar ET barriers with open-chain ligand (dien), but substantially increasing PT barriers.
Studying and understanding the fragmentations of M?+ species is fundamentally important and a formidable challenge—both charge-directed and radical-driven fragmentations play important roles, in a competitive manner, in the dissociations of M?+ species. Chapters 3.2-3.4 were built upon successful gas phase syntheses of a wide variety of M?+ species.
Chapter 3.2 reports the novel Cβ–Cγ bond cleavage of tryptophan residues in the dissociations of various tryptophan-containing M?+ species, resulting in a neutral 116-Da loss; this process is an α-radical–induced fragmentation. Substitution of the tryptophan residue by a 1-methyltryptophan residue revealed that the 116-Da neutral species is a radical with an unpaired electron on the indole nitrogen atom. Chapter 3.3 describes a systematic examination of tryptophan-containing model systems, both with and without basic residues, to unveil the mechanisms of Cβ–Cγ bond cleavages. M?+ species containing non-basic residues undergo protonation of the γ-carbon atom of the tryptophan residue, thereby weakening the Cβ–Cγ bond and facilitating its cleavage. The formation of [1H-indole]?+ (m/z 117) or [M – CO2 – 116]+ ions is a competition between two incipient fragments for the proton in a dissociating proton-bound dimer. In basic residue containing M?+ species, the proton is tightly sequestered by the basic side chain, resulting in more accessible radical migration barriers prior to subsequent bond cleavages; DFT calculations supported the notion that the charge-remote radical-driven pathway is more favorable than the proton-driven process by 6.2 kcal/mol.
Selective radical-induced fragmentations were then used to investigate the radical propagation processes occurring via hydrogen atom transfers—in particular, for Cα–C bond cleavages leading to the formation of an+ ions. The energetics and kinetics of the dissociations of [RGn–2FG7–n – CO2]?+ (n = 2–6) with well-defined C-terminal α-radicals were determined by RRKM modeling of surface-induced dissociation experiments and DFT calculations, revealing that radical propagations in peptide radical cations are not necessarily stepwise processes. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Formation, isomerization and dissociation of radical cationicpeptidesNg, Chun-ming, Dominic., 伍俊明. January 2011 (has links)
A fundamental understanding of the isomerization and fragmentation of peptide ions forms the scientific basis underlying peptide sequencing in the gas phase—an important emerging analytical technique routinely used in proteomics applications. Gas phase dissociation of odd-electron radical peptide cations (M?+) provides an alternative and complementary analytical method for identifying peptide sequences; this fragmentation behavior is distinct from that of even-electron protonated peptides ([M+H]+). Despite recent experimental and theoretical advances in studies of radical cationic peptides, their gas phase chemistry remains poorly understood.
The first part of this Thesis documents three mechanistic studies on the formation, isomerization, and dissociation of prototypical tripeptide radical cations in the gas phase using biological mass spectrometry. A combination of low-energy collision-induced dissociation (CID) experiments and density functional theory calculations at the B3LYP 6-31++G(d,p) level of theory was used to investigate the influence of the position of the radical site and the basicity of the amino acid residues in the radical cationic tripeptide analogs on their dissociation pathways. The CID spectra of two isomeric glycylglycyltryptophan radical cations—[GGW]?+ and [G?GW]+, with well-defined initial radical sites at the 3-methylindole ring and the N-terminal α-carbon atom, respectively—are significantly different. The former leads to the formation of [a3 + H]?+, [c2 + 2H]?+, and [z1 – H]?+ product ions through C–Cα
and N–Cα peptide bond cleavages, while the latter leads to the predominant fragment ions of y1+, [b2 – H]?+, and [b3 – H]?+ via amide bond cleavages. After substitution of the central glycine residue of GGW with an arginine residue, however, the two isomers [G?RW]+ and [GRW]?+ produced almost identical CID spectra. The calculated energy barriers and microcanonical rate constants for isomerizations and competitive dissociations are in accordance with the perception that isomerizations between the GGW isomers could not compete with their fragmentations. For the radical cationic isomers, the presence of the highly basic arginine residue decreases the isomerization barriers (ca. 7–11 kcal/mol) and mediates facile hydrogen atom transfers—both along the peptide backbone and within the side chain residues—prior to subsequent
dissociations. The effect of a basic amino acid residue on the isomerizations and dissociations of α-carbon–centered radical peptides also extends to distinctive Cβ–Cγ bond cleavages of isobaric leucine and isoleucine (Xle) residues. The CID spectra of [G?RXle]+ radical cations lead to the formation of characteristic product ions resulting from losses of ?CH(CH3)2 in [G?RL]+ and ?CH2CH3 in [G?RI]+ through Cβ–Cγ side-chain cleavages of (iso)leucine residues, allowing the two peptides to be distinguished.
Finally, the first implementation of laser-induced dissociation (LID) on a
hybrid quadrupole linear ion trap mass spectrometer is presented. After laser
irradiation of mass-selected and -trapped ions in the quadrupole linear ion trap, LID spectra of [M+H]+ undergo both facile backbone and side-chain cleavages. These products are strikingly different from those formed in the CID spectra of [M+H]+, but are similar to those in the corresponding CID spectra of M?+. This approach provides an alternative means of identifying peptide sequence in shogun proteomic analysis. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Dissociation and characterization of cationic radical peptidesXu, Minjie, 许敏洁 January 2013 (has links)
Gas phase fragmentations of cationic radical peptides provide important fundamental information that forms the basis for peptide sequencing by using mass spectrometry. Presenting results from low-energy collision-induced dissociation (CID) experiments and theoretical density functional theory (DFT) calculations in conjunction with Rice–Ramsperger–Kassel–Marcus modeling, this thesis describes some of the chemical properties, including the locations of the charge and radical sites that determine the gas-phase chemistry of peptide radical cations.
The first Section (3.1) documents the dissociations of two isomeric glycylglycylarginine methyl ester radical cations, [G•GR–OMe]+ and [GG•R–OMe]+, with well-defined initial radical sites at the N-terminal and middle α-carbon atoms, respectively. These two isomers undergo similar fragmentations to form the y2+ ion and protonated allylguanidine; their identical CID spectra suggest that isomerization occurs prior to dissociation. DFT calculations at the B3LYP/6-31++G(d,p) level revealed that the proton is sequestered on the guanidine group of the side chain in the presence of a highly basic arginine residue, thereby decreasing the isomerization barriers among the α-carbon–centered radicals to approximately 36 kcal mol–1 (cf. 45 kcal mol–1 for the non-basic [GGG]•+ analogues) and facilitating the radical migration along the peptide backbone and subsequent dissociation reactions.
The second section (3.2) describes an investigation into the specific effect of the N-terminal basic residue on selective Cα–C bond cleavage of aromatic-containing radical cationic peptides. Upon replacing the arginine residue of [R(G)n–2X(G)7–n]•+ by a less-basic lysine residue, forming [K(G)n–2X(G)7–n]•+ (X = Phe or Tyr; n = 2–7) analogues, the selective Cα–C peptide bond cleavage no longer occurs. The dissociations of the prototypical radical cationic tripeptides [RFG]•+ and [KFG]•+ at the second Cα–C peptide bonds of α-radical intermediates proceed with comparable barriers (ca. 33 and 35 kcal mol–1, respectively); the generation of the competitive [b2 – H]•+ fragment from [RFG]•+ (ca. 40 kcal mol–1) is much higher in energy than that from [KFG]•+ (ca. 27 kcal mol–1). Thus, the selective Cα–C bond cleavage product from [KFG]•+ can be overridden by the [b2 – H]•+ species in the absence of a basic N-terminal residue.
Section (3.3) further examines the mechanistic roles of various α- andβ-carbon–centered radicals prior to Cα–C bond cleavage, leading to the observation of novel x-type radical fragments. DFT calculations and RRKM modeling of a prototypical π-radical cationic system, [AY]•+, suggested that direct Cα–C bond cleavage leading to the formation of the [x1 + H]•+ species is thermodynamically comparable (ca. 16 kcal mol–1) with, but kinetically at least three-fold more favorable than, the well-characterized competitive formation of [c1 + 2H]+ and [z1 – H]•+ species. This finding agrees well with the experimental yield of the [x1 + H]•+ radical cation being higher than that of the minor [c1 + 2H]+ species. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Immobilized peptides as high affinity capture reagents for multimeric proteins and structural studies of cell-targeting peptidesNaffin, Jacqueline Lara 10 June 2011 (has links)
Not available / text
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The characterisation and fragmentation of peptides by mass spectrometry / by Adrian Morley Bradford.Bradford, Adrian Morley January 1996 (has links)
Copies of author's previously published articles inserted. / Bibliography: leaves 191-213. / xiv, 213 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 1997
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Biological mass spectrometry of peptides and glycopeptidesSiu, Shiu-on., 蕭紹安. January 2008 (has links)
published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Investigation of the effect of the precursor ion heterogeneity on the fragmentation of the model peptides under electron capture dissociation.January 2011 (has links)
Chen, Fan. / "October 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 85-91). / Abstracts in English and Chinese. / ABSTRACT --- p.III / 摘要 --- p.IV / ACKNOWLEDGENTS --- p.V / TABLE OF CONTENTS --- p.VI / LIST OF FIGURES --- p.VIII / LIST OF TABLES --- p.X / SYMBOLS AND ABBREVIATIONS --- p.XI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Mass spectrometry in proteomics --- p.1 / Chapter 1.2 --- Fourier-transform ion cyclotron resonance mass spectrometry --- p.2 / Chapter 1.2.1 --- Introduction --- p.2 / Chapter 1.2.2 --- Ionization --- p.3 / Chapter 1.2.3 --- Ions in ICR --- p.4 / Chapter 1.2.4 --- Ions excitation and detection --- p.7 / Chapter 1.3 --- Tandem mass spectrometry --- p.8 / Chapter 1.4 --- Electron capture dissociation --- p.12 / Chapter 1.4.1 --- Features ofECD --- p.13 / Chapter 1.4.2 --- Two popular mechanisms for ECD --- p.14 / Chapter 1.4.2.1 --- The Cornell mechanism --- p.15 / Chapter 1.4.2.2 --- The Utah-Washington mechanism --- p.17 / Chapter 1.4.3 --- Recombination energy --- p.19 / Chapter 1.5 --- Outline of the present work --- p.21 / Chapter CHAPTER 2 --- INSTRUMENTATION AND EXPERIMENTAL --- p.22 / Chapter 2.1 --- Fourier-transform ion cyclotron resonance mass spectrometer --- p.22 / Chapter 2.1.1 --- Vacuum system --- p.24 / Chapter 2.1.2 --- Nanospray system --- p.26 / Chapter 2.1.3 --- Ion transfer system --- p.29 / Chapter 2.1.4 --- Infinity´ёØ cell --- p.29 / Chapter 2.1.5 --- Electron emission source --- p.31 / Chapter 2.1.6 --- Data acquisition system --- p.32 / Chapter 2.2 --- Experimental --- p.32 / Chapter 2.2.1 --- Simple ESI acquisition pulse program --- p.32 / Chapter 2.2.2 --- ESI-ECD acquisition pulse program --- p.35 / Chapter CHAPTER 3 --- FRAGMENTATION OF MODEL PEPTIDE IONS IN DIFFERENT CHARGE STATES UNDER ECD CONDITIONS --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2. --- Experimental --- p.41 / Chapter 3.2.1 --- Sequence design and sample preparation --- p.41 / Chapter 3.2.2 --- ECD under fourier-transform ion cyclotron mass spectrometer --- p.43 / Chapter 3.3 --- Results and discussion --- p.43 / Chapter 3.3.1 --- General features of ECD spectra --- p.43 / Chapter 3.3.1.1 --- ECD of RRRR --- p.43 / Chapter 3.3.1.2 --- ECD of KKKK --- p.47 / Chapter 3.3.2 --- Effect of charge state of precursor ions --- p.49 / Chapter 3.3.3 --- Effect of proton carriers --- p.52 / Chapter 3.3.4 --- Effect of proton carrier location --- p.54 / Chapter 3.4 --- Conclusions --- p.60 / Chapter CHAPTER 4 --- EFFECT OF PRECURSOR ION HETEROGENEITY ON ECD FRAGMENTATION --- p.62 / Chapter 4.1 --- Introduction --- p.62 / Chapter 4.2 --- Method --- p.63 / Chapter 4.2.1 --- Preferential dissociation index --- p.64 / Chapter 4.2.2 --- Precursor ion heterogeneity --- p.65 / Chapter 4.3 --- Results and discussion --- p.67 / Chapter 4.3.1 --- PDI in model peptides --- p.67 / Chapter 4.3.2 --- PIH and PDI in RRRR and KKKK --- p.71 / Chapter 4.3.3 --- PDI and PIH in two-lysine containing peptides --- p.73 / Chapter 4.3.4 --- PDI and PIH in other peptides --- p.80 / Chapter 4.4 --- Conclusions --- p.82 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.83 / References --- p.85 / Chapter Appendix I: --- Pulse program for simple MS and MS/MS experiment --- p.92 / Chapter (A) --- Simple ESI FT-ICR MS experiment --- p.92 / Chapter (B) --- ESI ECD FT-ICR MS experiment --- p.95 / Chapter Appendix II: --- ECD spectra of AC-XAAAXAAAXAAAX-NH2 peptide series in different charge states --- p.99 / Chapter Appendix III: --- The PDI of the hypothetic peptide --- p.110 / Chapter Appendix IV: --- The PIH among the investigated system --- p.111
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Identification and characterization of vasotocin and mesotocin peptides and receptorsSearcy, Brian T. 09 December 2004 (has links)
The neurohypophysial peptide system is involved in modulating a
variety of physiological, neurological, and behavioral responses in
vertebrates. The principal forms of these peptides in non-mammalian
tetrapods are vasotocin (VT) and mesotocin (MT). The studies described
in this thesis used pharmacological, molecular, and biochemical
techniques, along with phylogenetic analyses, to identify and characterize
the mRNA sequences encoding the neurohypophysial peptide precursor
proteins and their receptors in urodele amphibians.
The cDNAs encoding preproVT and preproMT were amplified by
PCR from the brains of two salamander species; the rough-skinned newt,
Taricha granulosa, and the red-legged salamander, Plethodon shermani.
The neurohypophysial peptides encoded by the identified Taricha cDNAs
were VT and MT; the Plethodon cDNAs encoded VT and a novel MT-like
peptide, [Val⁴]-MT. Phylogenetic analyses grouped both the Taricha and
Plethodon preproVT and preproMT-like sequences with previously
identified tetrapod preproVT-like and preproMT-like sequences,
respectively. Additional analysis of the preproneurohypophysial sequences
indicated that gene conversion (non-homologous crossing over of DNA
sequences) appears to have occurred more frequently in mammals than in
other tetrapod classes.
The cDNAs encoding the VT receptor (VTR) and MT receptor (MTR)
were amplified from the brain of T. granulosa by PCR. Sequence identity,
and phylogenetic analysis, indicated that the Taricha MTR and VTR were
most similar to MTR/OTRs and V[subscript 1a]-like VTRs, respectively. Distribution of
PCR amplicons specific to the Taricha MTR and VTR matched previously
reported tissue distributions of MTRs and VTRs in other vertebrates in
every tissue but kidney, from which the Taricha primers were unable to
amplify a cDNA product. Binding experiments of transiently expressed
Taricha MTR indicated two binding states, and allowed the determination
of ligand binding affinities for this receptor. Inositol phosphate
accumulation assays demonstrated that the expressed Taricha MTR and
VTR cDNA produced functional receptors, and allowed calculation of ligand
potencies of activation and inhibition. Surprisingly, an antagonist
frequently used in behavioral experiments to specifically block VTR activity,
inhibited inositol phosphate accumulation in cells transfected with either the
Taricha MTR or VTR. In conclusion, these studies report the first identified cDNA
sequences encoding the preproVT, preproMT, MTR, and V[subscript 1a]-like VTR
proteins from urodele amphibians. / Graduation date: 2005
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The observation of evolutionary trends in amphibians and the analysis of negative ion fragmentations in large peptide systems by mass spectrometry / by Simon Todd Steinborner.Steinborner, Simon Todd January 1997 (has links)
Copies of author's previously published articles inserted. / Bibliography: leaves 195-196. / xv, 196 leaves : ill., maps ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this research is to observe the evolutionary relationships within and between frog species, as well as to discover potentially useful medicinal peptides. The two main areas of research of this thesis are the characterisation of peptides from frogs belonging to the genus Litoria and the analysis of negative ion fragmentations from large peptides. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 1997?
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