Structural Characterization of Gas-Phase Biomolecular Ions with Novel Mass Spectrometry and Computational Methods

Abstract

This dissertation presents the structural characterization of gas-phase biomolecular ions through novel mass spectrometry-based techniques and computational studies. Ions were generated via electrospray ionization and analyzed using tandem mass spectrometry (MSn) within an ion trap mass analyzer. Fragmentation methods used were collision-induced dissociation (CID) and ultraviolet photodissociation (UVPD) at various wavelengths. Cyclic ion mobility experiments provided experimental collision cross sections (CCS), revealing three-dimensional shapes of ions. UV-Vis action spectroscopy was used to study the electronic structures of ions. Born-Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) were used to obtain low-Gibbs-energy structures with theoretical CCS and vibronic absorption spectra. Background for these experimental and theoretical methods is discussed in Chapter 1. Chapter 2 and Chapter 3 focus on the resolution of identity in dissociations in DNA codon cations and cations radicals. Mono- and diprotonated cations were generated by electrospray ionization. Cation radicals were generated by electron transfer (ET). Dissociations in CID-MS included nucleobase loss and backbone cleavage. Nucleobase loss was found to be highly dependent on both the type and position of the nucleobase within the DNA codon. The w2+ ion formation was universal in dissociations of cations while the d2+ ions were dominant in that of cation radicals. In both cases 15N-labeling in nucleobases was used to resolve dissociations in symmetric sequences dXXX and dXYX. Plausible dissociation pathways were proposed based on low-Gibbs-energy structures of isomers and conformers. Chapter 4 investigates the secondary structure transitions of DNA from solution to the gas phase. Cyclic ion mobility was employed to study the dGCGAAAGC heptanucleotide hairpin and its sequence-scrambled isomers. The 2+, 3+, and 4+ ions exhibited mixtures of protomers and conformers, as evidenced by multiple peaks in ion mobility arrival time distributions. Low-energy structures revealed zwitterionic features and disruption of G···C Watson-Crick pairs in the gas-phase ions. The experimental and theoretical CCS for low-energy structures showed reasonable agreement. Chapter 5 describes the development of nitrile imine as a novel photo-cross-linker. Thermal and photodissociation of tetrazole-peptide conjugates generated –N2 ions that crosslinked intrinsic functional groups of peptides, producing unique fragments in CID-MS3. UV-Vis action spectroscopy and cyclic ion mobility, supported by comprehensive computational studies, was used to characterize the –N2 ions. Additionally, nitrile imines selectively crosslinked with guanine in peptide-dinucleotide complexes, highlighting their potential in detecting non-covalent interactions. Chapter 6 explores the interactions between the tetrazole photo-tag and the N-terminal Asp, Glu, Asn, and Gln residues. Nitrile imines underwent efficient crosslinking to the side-chain amides and carboxyl groups, as indicated by the characteristic ʋn type fragment ions and the matching between experimental and theoretical CCS. Other signature fragments were loss of phenylhydrazine and internal alanines. Chapter 7 further explores the effect of basic N-terminal residues Arg, Lys, and His on crosslinking. The proposed mechanism involved proton transfer-assisted nucleophilic attack and oxygen transfer. Chapter 8 summarizes all the work conducted and proposes future directions for exploration.