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Ion/ion Reactions in Primary Structure Interrogation of Intact Proteins by Mass Spectrometry

Duselis, Elizabeth
Thesis/Dissertation; Online
Duselis, Elizabeth
Hunt, Donald
This dissertation describes method development for intact protein characterization by mass spectrometry with ion/ion reactions. In this first project, novel methodology of sequential ion/ion reactions, electron transfer dissociation (ETD) followed by ion/ion proton transfer, is used on intact hemoglobin subunits in clinical samples to identify single amino acid substitutions. In this study, 22 variants were definitively identified, including a previously unreported hemoglobin variant now known as Hemoglobin Charlottesville. In the second portion of the dissertation, ion/ion reaction kinetics play a pivotal role. A novel method for investigating the physics of ion motion during an ion/ion reaction is presented. By applying a supplemental alternating current electric potential during the ion/ion reaction, resonant ions are indirectly evaluated through arrested reaction rates. This method uncovered alterations to ions’ fundamental secular frequencies in a quadrupole ion trap due to simultaneous confinement of both ion polarities. This knowledge builds the basis for development of parallel ion parking during ETD, an analytical method in which first-generation ETD fragments are resonantly excited. The arrested reaction rate of these ions lead to their preservation. The resulting sequence coverage of standard proteins 8.6 kDa ubiquitin, 17 kDa apomyoglobin, 20 kDa histone H1, and 21 kDa Protein G are 96%, 88%, 73%, and 80% respectively. The high incidence of complementary fragment ion pairs (93%, 78%, 85%, and 85% respectively) are promising for the future of intact protein interrogation.
University of Virginia, Department of Chemistry, PHD (Doctor of Philosophy), 2019
Published Date
PHD (Doctor of Philosophy)
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