Separation and structural characterization of pendular macromolecules using low-field differential ion mobility spectrometry and divergent field ion mobility focusing
type: CreativeWork
description: Ion mobility spectrometry coupled with mass spectrometry (IMS/MS) is a versatile platform for simplifying complex mixtures, separating isomers, and probing gas-phase structure. Yet extending IMS/MS to larger peptides and proteins requires greater resolving power and means of extracting structural information beyond orientational averaging of ion-molecule collision cross sections (ΩAvg). This dissertation develops and applies complementary linear and differential IMS methods for the analysis of large biomolecules, beginning with baseline separation of the largest known D/L peptide epimers, crustacean hyperglycemic hormone (CHH-A, 72 residues), by trapped IMS, cyclic IMS, and high-resolution FAIMS, which extends gas-phase epimer analysis into the protein size range and establishes differential collision-induced unfolding (CIU) as an additional means for distinguishing peptide epimers. Low-field differential IMS (LODIMS), which leverages dipole alignment, was advanced through the development of near-ideal fully flexible rectangular waveforms that expand the available separation space and permit more accurate determination of key structural descriptors such as directional collision cross sections (ΩDir) and dipole moments. Integration of this LODIMS platform with Orbitrap MS and MS/MS for top-down analysis of Concanavalin A (ConA, 25.6 kDa) revealed new proteoforms and demonstrated that LODIMS can align smaller proteins while providing access to their directional cross sections and weaker dipole moments. Finally, this work introduces divergent field ion mobility focusing (DIFIMOF), a fundamentally new IMS separation which separates aligned macromolecules in inhomogeneous fields according to the super vs. sublinear scaling of their EC(ED) curvature. This enables the fractionation of macroions into two distinct subpopulations based on the applied waveform polarity, providing a mode of separation orthogonal to LODIMS, FAIMS, and linear IMS.
abstract: Ion mobility spectrometry coupled with mass spectrometry (IMS/MS) is a versatile platform for simplifying complex mixtures, separating isomers, and probing gas-phase structure. Yet extending IMS/MS to larger peptides and proteins requires greater resolving power and means of extracting structural information beyond orientational averaging of ion-molecule collision cross sections (ΩAvg). This dissertation develops and applies complementary linear and differential IMS methods for the analysis of large biomolecules, beginning with baseline separation of the largest known D/L peptide epimers, crustacean hyperglycemic hormone (CHH-A, 72 residues), by trapped IMS, cyclic IMS, and high-resolution FAIMS, which extends gas-phase epimer analysis into the protein size range and establishes differential collision-induced unfolding (CIU) as an additional means for distinguishing peptide epimers. Low-field differential IMS (LODIMS), which leverages dipole alignment, was advanced through the development of near-ideal fully flexible rectangular waveforms that expand the available separation space and permit more accurate determination of key structural descriptors such as directional collision cross sections (ΩDir) and dipole moments. Integration of this LODIMS platform with Orbitrap MS and MS/MS for top-down analysis of Concanavalin A (ConA, 25.6 kDa) revealed new proteoforms and demonstrated that LODIMS can align smaller proteins while providing access to their directional cross sections and weaker dipole moments. Finally, this work introduces divergent field ion mobility focusing (DIFIMOF), a fundamentally new IMS separation which separates aligned macromolecules in inhomogeneous fields according to the super vs. sublinear scaling of their EC(ED) curvature. This enables the fractionation of macroions into two distinct subpopulations based on the applied waveform polarity, providing a mode of separation orthogonal to LODIMS, FAIMS, and linear IMS.
author: Thurman, Hayden A.
date published: 2026-05
date modified: 2026-07-24T06:06:34Z
provider: Wichita State University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Wichita State University