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

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.