Structural Characterization of Proteins, Peptides and Oligosaccharides Based on Radical Chemistry and Mass Spectrometry

Mass spectrometry (MS) is best known for measuring molecular weight and probing the chemical structure. Radical directed dissociation (RDD) is a tandem MS fragmentation method for hydrogen deficient radical precursors, which provides complementary structure information compared to other tandem MS methods. Fragmentation pathways of peptide radical isomers in collision induced dissociation are radical site dependent. The purpose of my first project is to use RDD fragmentation patterns from peptide radical isomers as probes of peptides gas phase structure and conformation flexibility. Radicals are site-specifically generated by photodissociation of carbon-iodine bonds at ortho-, meta-, or para- position of a benzoyl ring labeled at peptide amine group. Radicals at these sites have almost identical bond dissociation energies and thus the only difference comes from the steric effect. We utilized the steric effect and isotope deuterium labeling to study peptide gas phase structure and its influence on intramolecular radical migration. Evaluating protein structure in the gas phase is useful for understanding the intrinsic forces which influence protein folding and for determining the feasibility of probing condensed phase structure with gas phase interrogation. KIX is a three-helix bundle protein that has been reported previously to preserve the condensed phase structure in the gas phase. In another project, structure dependent RDD is used to examine the gas phase structure of KIX by establishing residue specific distance constraints which can be used to assess candidate structures obtained from molecular dynamics simulations. The data obtained by RDD is consistent with KIX structures that largely retain condensed phase structure as determined previously by NMR. A new method is developed to probe peptide structure in vacuo utilizing di-radical recombination reaction. Commonly, the pairing of electrons which occurs when two radicals interact is a radical exothermic process without an activation barrier. In the case of a separated peptide ion containing two radicals in the gas phase, such radical coupling reaction is an intramolecular cyclization process and generates a cyclic peptide. Therefore, examining the sites where the di-radical recombination occurs can provide local contact information. Herein, two radical precursors are site specifically introduced to polyproline at the N and C terminal residue, respectively. Internal fragments from cyclized polyproline peptides and ion-molecule reaction data indicate that polyprolines no longer adopts a helical structure as in the solution which would prevent diradical interaction. Intermolecular homolytic aromatic substitution (HAS) reaction between benzoate phenyl radical and aromatic side chains in large peptide is observed and investigated. Compared to those radical precursors we previously used, 2-iodo benzoic acid has a much higher propensity to add to the aromatic rings in peptides. HAS is in competitive with radical migration reaction and the product branching ratio depends on specific complex structure. The reaction mechanisms are verified by labeling at various key positions for tryptophan and tyrosine. Novel side chain losses from aromatic residues (-116W, -93Y, -77F) are rationalized by HAS. Phenylalanine is a chemically inert residue. Such unique side chain loss (i.e., -77F) allows identification of phenylalanine residue in peptides. Study of phenyl radical damage to oligopeptides in the gas phase by radical substitution reaction is achieved for the first time. New methods are developed to dissociate oligosaccharides based on RDD. Radical saccharides can be generated by either intermolecular radical migrations or covalent labeling with radical precursors. Compared to CID of even electron saccharides, RDD provides rich fragmentations including cross-ring cleavages. Positional isomers of oligosaccharides (e.g., lacto-N-fucopentaose isomers and lacto-N-difucohexaose isomers) are able to be distinguished by RDD owing to observed signature fragments