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

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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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1972. / Bibliography: leaves [192]-206. / xvi, 206 l illus., tables