Semester

Spring

Date of Graduation

2021

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Glen P. Jackson

Committee Member

Stephen J. Valentine

Committee Member

Jonathan W. Boyd

Committee Member

Justin A. Legleiter

Committee Member

Danyel H. Tacker

Abstract

The recent surge in the analysis of biomolecules has driven the need for robust, versatile, sensitive, and accurate techniques such as tandem mass spectrometry for compound structural elucidation. Many fragmentation techniques have been developed over the years, but each has their own limitations, such as the loss of post-translational modifications (PTMs), inability to fragment singly charged precursor ions or cost prohibitiveness. To help fill this gap, a novel fragmentation technique called charge transfer dissociation (CTD) has been developed to fragment stored precursor ions through radical-driven fragmentation processes using kiloelectronvolt reagent cations. CTD has shown success in the analysis of proteins, peptides, lipids and oligosaccharides but, until now, the underlying principles behind this fragmentation technique have not been thoroughly explored. This dissertation, therefore, focused on the fundamentals of CTD efficiencies and mechanisms and the extent of structural information that can be gained depending on the charge state and charging adducts of different precursor ions like peptides and oligosaccharides.

The first set of experiments investigated the identity of the reagent gas used in CTD and the impact on the sequence coverage and fragmentation efficiency for the analysis of a model peptide, bradykinin and a model oligosaccharide, κ-carrageenan with a degree of polymerization of four (dp4). In past work, CTD employed helium as a reagent gas, but due to the increased scarcity and expense of helium as a consumable, this work explored a variety of alternative reagent gases, including Ar, H2, He, N2, O2, and lab air. Initially, CTD was contrasted with low-energy collision-induced dissociation (LE-CID) for both bradykinin and κ-carrageenan dp4. All of the CTD reagents gases generated near-complete sequence coverage of bradykinin and LE-CID only generated ~56% sequence coverage. For analysis of κ-carrageenan dp4, all the CTD reagents gases generated more structural information than CID, and CTD preserved labile sulfate groups while providing cross-ring cleavages. In contrast, LE-CID spectra contained sulfate losses, glycosidic cleavages and neutral losses. All five reagent gases generated consistent sequence coverage and fragmentation efficiencies relative to He-CTD, which suggests that the ionization energy of the reagent gas has minimal impact on the fragmentation of the biological ions. The majority of the activation energy for bradykinin and κ-carrageenan dp4 comes from the electron stopping mechanism, which involves long-range coupling of reagent cations and electrons bound in the highest occupied molecular orbitals (HOMOs) of the biological ions. Based on these results, any of the alternative reagent gases tested can function as effective options for CTD experiments.

He-CTD and LE-CID were then used to analyze alkali and alkaline earth metal adducts of a branched glycan, XXXG, to determine if metal adducts would impact the fragmentation patterns and structural characterization of the analyte. Both singly and doubly charged precursors were analyzed and they included H+, Na+, K+, Ca2+ or Mg2+ cations. The LE-CID spectra were dominated by glycosidic cleavages and numerous neutral losses which complicated spectral interpretation. Irrespective of the metal adduct, He-CTD generated abundant and numerous glycosidic and cross-ring cleavages that were structurally informative and able to identify the 1,4-linkage and 1,6-branching pattern of XXXG. Surprisingly, both LE-CID and He-CTD generated singly charged product ions from doubly charged adducts of calcium and magnesium. A similar phenomenon is observed with ECD of magnesium and calcium adducts and is due to the loss of H+ from the metalated product ions and the formation of a protonated complementary product ion. However, during He-CTD, the [M+Mg]2+ precursor generated more singly charged product ions than [M+Ca]2+ presumably because Mg has a higher second ionization potential than Ca. Overall, even though the metal adducts altered the m/z values of the product ions, the metal adducts did not inhibit the generation of structurally informative product ions and the identification of the 1,4-linkage and 1,6-branching pattern of XXXG with He-CTD.

The final set of experiments examined if He-CTD could effectively characterize the structure of a mannuronic acid oligomer and how sodium-hydrogen exchanges impact the fragmentation pathways of polymannuronic acid. He-CTD was able to successfully identify the 1,4-linkage pattern between the core mannuronic acid residues of the [M+Na]+ and [M+2Na]2+ precursors using multiple product ions including, 3,5An, 2,4Xn, 1,4A3 and 2,4An cross-ring cleavages. Upon cursory inspection of the [M+3Na-H]2+ and the [M+3Na-2H]+ product ion maps, the number of cross-ring cleavages observed appeared to increase as the number of sodium-hydrogen exchanges increased. However, the pattern did not continue when the [M+4Na-3H]+, [M+5Na-3H]2+ and [M+6Na-4H]2+ were investigated because there was a decrease in the number of cross-ring fragments generated between the [M+5Na-3H]2+ and [M+6Na-4H]2+ precursors. In EDD of glycosaminoglycans, an analogous situation occurs and is attributed to the possible involvement of carboxyl hydrogens in the generation of cross-ring cleavages through hydrogen rearrangements. The main difference between the spectra with and without sodium-hydrogen exchange was the increased number of ambiguous product ions generated in spectra with sodium-hydrogen exchanges. The 18O labeling on the reducing end helped lessen the number of ambiguous product ions generated, but there were numerous isobaric product ions generated due to the possible product ions from the sodium-hydrogen exchanges. Since comparable structural information can be gathered without sodium-hydrogen exchange, precursors without sodium-hydrogen exchange should be targeted until He-CTD is coupled to high-resolution mass spectrometry to differentiate the isobaric product ions.

Embargo Reason

Publication Pending

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