Date of Graduation

2017

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Stephen J Valentine

Committee Co-Chair

Peter M Gannett

Committee Member

Lisa A Holland

Committee Member

Glen P Jackson

Committee Member

John B Mertz

Abstract

Over the last few decades, the widespread application of soft ionization techniques such as electrospray ionization (ESI) coupled with mass spectrometry (MS) facilitated the characterization of large biomolecules in the gas phase. Under suitable conditions and during the short timescale of the desolvation process associated with ESI (10-12-102 s), the solvent-free protein ions preserve significant portions of their native solution-phase structure and are presumed to be kinetically protected from thermodynamic destabilization such as unfolding-refolding processes occurring in the gas phase that could yield an "inside-out" structure.;The combination of ESI with ion mobility spectrometry- mass spectrometry (IMS-MS) provides information regarding higher-order structure of proteins and their complexes. IMS-MS has the ability to resolve and probe conformers of protein ions of a particular charge state based on their mobility through an inert buffer gas in drift tube under the influence of a constant electric field. An ion's mobility is dependent upon its charge and shape and can be used to determine the orientationally-averaged collision cross section (CCS) of the ion with the buffer gas. Comparison of experimentally measured CCS values with theoretical CCS numbers obtained from a series of computer-assisted molecular dynamics simulations (MD) which explore the conformational space of protein ions provides insight into candidate structures for each conformer.;Although, CCS values provide a rough estimate of the overall shape of different molecules, IMS-MS alone cannot distinguish three dimensional structures of a series of conformers that represent the same mobility and thus would arrive at the same time at the exit region of the drift tube. The combination of IMS-MS with hydrogen-deuterium exchange (HDX) experiments as a gas-phase chemical probe of structure provides the opportunity to distinguish among these conformer types.;In this work, the gas-phase ion conformers of a model peptide have been studied through CCS measurements, HDX behavior analysis and extensive (MD) simulations.;Initially, an advanced protocol is introduced in order to achieve an impartial sampling of phase space targeting both higher-energy and more thermodynamically-stable structures. The gas-phase transport properties of the ion conformers - including their dynamics at experimental temperatures- have been monitored, and, combined with an optimized clustering and data mining method, accurate CCS determination has been accomplished. These data provided the first criterion to filter through a substantial pool of conformations in order to obtain a series of candidate structures (CCS matched) with significant structural variation.;A hydrogen accessibility scoring (HAS)-number of effective collisions (NEC) model is applied to the candidate structures obtained from MD simulations. The HAS-NEC model produced hypothetical, per-residue deuterium uptake values. This information then provided the overall structural contribution from each in-silico structure leading to the best match to experimental results. The comparison of predicted and experimentally observed isotopic envelopes of various mass spectral fragment ions supported the accuracy of the model. With these results, the hypothetical HDX data were employed as a second dimension to narrow the sampled phase space and, together with the accurate CCS values, 13 nominal conformers with specific population contributions to the gas-phase ions were selected.;In the final installment of this work, extensive simulations of the ESI process were performed to monitor the behavior of the peptide ion, charge carriers and the droplets involved in the ionization process. The results provided a series of structures that match the nominal conformers obtained through CCS calculations and the HAS-NEC model. This method validation confirmed the accuracy of the HAS-NEC model in successfully predicting the representative gas-phase structures on a computationally-affordable timescale.

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