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Gas Phase Radical Migration in Peptides and Proteins


Radical chemistry represents a powerful set of reactions in which unstable odd electron species can undergo rapid and spontaneous chemical rearrangements. These reactions have recently found analytical use in the dissociation of peptides and proteins in mass spectrometry. In this dissertation, the factors controlling radical migration and dissociation are explored in detail through examination of model peptides and proteins by mass spectrometry and quantum mechanical calculation. Understanding the behavior of radicals in proteins is key to being able to further enhance their analytical usefulness.

Radicals already play an important role in electron capture dissociation (ECD) and electron transfer dissociation (ETD) mass spectrometry. When applied to peptides both gas phase dissociation methods yield nearly complete and uniform sequence coverage while retaining labile post-translational modifications (PTMs). In contrast, a recently developed technique, radical directed dissociation (RDD), induces fragmentation at very specific locations in derivatized peptides and proteins which has proven useful in a variety of applications including the facile identification of PTMs. RDD provides very specific fragmentation and is complimentary to the broad uniform fragmentation provided by ECD/ETD. Despite these differences in observed dissociation patterns, ECD, ETD, and RDD are all driven by radical chemistry. This disparity in observed fragmentation can be explained by radical conversion and migration.

Radical migration in peptides typically occurs via hydrogen atom abstraction. In this manner radicals may travel to different sites within a molecule assuming the intermediate hydrogen atom transfer reactions are favorable both thermodynamically and kinetically. In this work, the thermodynamic factors, specifically the X-H bond dissociation energies (BDEs) of the 20 canonical amino acids, are determined by quantum mechanical calculation. These BDEs are then used to predict radical induced dissociation in peptides. UV-initiated gas phase peptide radicals are then used to examine radical migration experimentally. Radical induced fragmentation is observed at locations far from the initial radical site clearly indicating that radical migration occurs. Furthermore, good correlation exists between the BDE predicted and experimentally observed radical migration patterns, regardless of charge polarity. It is also shown that radical migration pathways that do not yield immediate dissociation products can be tracked via ion-molecule reactions, specifically the reaction between a carbon centered radical and molecular oxygen gas.

The potential uses of radical chemistry in the examination of complex biomolecules such as proteins by mass spectrometry are numerous. The key to harnessing radicals for future analytical use lies in developing a deeper understanding of the thermodynamics and kinetics that control their behavior.

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