Gaseous Studies of Ionic Chromophores and Peptide Cation Radicals Generated from Electron Transfer

Thesis (Ph.D.)--University of Washington, 2016-03University of Washington Abstract Gaseous Studies of Ionic Chromophores and of Peptide Cation Radicals Generated from Electron Transfer Robert H. Pepin Chair of the Supervisory Committee: Professor František Tureček Department of Chemistry Electron transfer to multiply protonated peptide cations from anion radical donors in the gas phase has become an increasingly popular method for peptide and protein sequence analysis. Electron Transfer Dissociation (ETD) of peptides generate energetic cation radical species which usually undergo non-specific N-Cα bond dissociation to generate N-terminal c fragments and C-terminal z˙ radical fragments. The mechanism by which this N-Cα bond dissociation occurs is not yet fully understood. With ETD’s ability to preserve labile post translational modifications and thereby aid in the localization of modified amino acid residues it becomes easier for clinicians to understand abnormalities in proteoforms and thus more accurately diagnose and treat cancers. Given the potential for personalized medicine afforded by proteomics it is certainly a worthy goal to further our understanding by which the ETD fragmentation process works. Theoretical and experimental investigations reported herein are all targeted towards elucidating ETD fragmentation mechanistic details be it conformational effects, energy distribution or electronic structure of peptide cation radicals in the gas phase. Theoretical modeling of model systems has suggested several aspects of the fragmentation mechanism for electron based fragmentation methods, such as ETD. Calculations indicate that in the presence of a remote charge amide groups can capture electrons and become superbases, abstracting protons from even the highly basic arginine guanidinium group and facilitating formation of labile aminoketyl radicals leading to N-Cα bond dissociations. A thorough computational and experimental study of the ETD behavior of a relatively simple Gly-Leu-Gly-Gly-Lys pentapeptide is presented wherein electron attachment at various amide groups along the peptide backbone are modeled and shown to lead to the formation of the ETD products experimentally observed. Electron Capture Dissociation (ECD), a fragmentation method related to ETD, at very cold temperatures has been shown to have a reduced number of backbone cleavages when compared to ambient temperature measurements. This demonstrates the importance of peptide ion conformation in the gas phase when activated by electrons. In a study of a series of heptapeptide ions designed to have tightly folded conformations in the gas phase, it was found that low energy conformer searching, subsequent collision cross section calculations and experimentally derived cross sections are insufficient to distinguish between low energy peptide ion conformers on their own and that an additional structure probing method was needed. ETD fragmentation of these ions did show interesting effects that can be ascribed to the tightly compact structures the ions were designed with. A defining aspect of the initially proposed mechanism for ECD was that fragmentations occurred before energy redistribution and randomization; that is, ECD was considered to be a non-ergodic process. We present an experimental study utilizing the secondary radical-induced dissociation of the leucine side chain as a “thermometer” for the energies involved in the ETD process and arrive at the conclusion that ETD must be an ergodic process and that the neutral electron donor molecule formed by the ETD reaction cannot be formed in an excited electronic state. In a further attempt to probe the 3-D conformations of peptide ions in the gas phase, we are exploring the use of diazirine-carbene insertion photochemistry as a way of freezing gas phase conformations of peptide ions and interrogating these ions via various fragmentation methods in an attempt to locate the correct predicted ion conformations. We present here a study of a newly developed amino acid, photo-lysine, which is tagged with a diazirine chromophore for carbene insertion reactions. In order to more fully understand the factors which lead to a successful bond insertion reaction, non-covalent complexes of proton bound peptide dimers are formed via electrospray ionization, covalently “stitched” together and then analyzed for trends involving stitching efficiency and localization of the insertion point. Finally, initial ion absorbance action spectroscopy experiments are reported. Knowledge of the gas phase absorbance behaviors of charge reduced peptide cation radicals and studies of the z˙ radical fragments both generated via electron transfer will be useful in probing the electronic structures of these ions and thus the mechanistic details of ETD in more depth. Presented here is the action spectroscopy of two common organic dye molecular ions and a study of the absorbance bands of the diazirine chromophore in the newly synthesized photo-lysine amino acid as a prelude to peptide cation radical studies