Semester

Fall

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

Lisa A. Holland

Committee Member

Peng Li

Committee Member

Shikha Sharma

Abstract

The ever-changing landscape of mass spectrometry is driven by gaps in knowledge, and one of the biggest driving forces is the desire to understand the structure and dynamics of biomolecules in living organisms. New and better methods of tandem mass spectrometry are often at the heart of technological developments. Some fragmentation methods are limited to expensive FTICR instruments, some techniques are limited multiply charged precursors, and others are limited to breaking only the weakest bonds in precursor ions; all of which support the pursuit of new activation methods. Charge transfer dissociation (CTD) addresses some of these concerns by providing structurally informative fragmentation of singly or multiply charged precursors using a benchtop instrument. Since its development, CTD has been successfully applied to several classes of molecules including peptides, lipids, and oligosaccharides. This work builds on those foundations to address three areas of potential interest: 1) the characterization of macrocyclic structures like antibacterial macrolides, 2) the differentiation of leucine and isoleucine residues in peptides, and 3) the discrimination of amino acid epimers and isomers, like D- and L forms of aspartic acid and isoaspartic acid in different peptides.

For macrocyclic structures, we applied CTD to a variety of natural and synthetic macrocycles, including cobalamins (e.g., Vitamin B12), macrolides (e.g., Erythromycin) and a synthetic polymer (e.g., cyclic Nylon-6,6). For vitamin B12, CTD generated rich spectra that contained a variety of cleavages around the nucleotide loop but not within the corrin ring of vitamin B12. CTD of vitamin B12 also provided several neutral losses that corresponded to the axial ligand plus either the central cobalt or an acetamide neutral, which have the same nominal mass. The resolution of the 3D ion trap was insufficient to resolve the elementally distinct product ions, so some peak assignments are currently ambiguous. For Erythromycin, we again observed more numerous fragments with CTD than with CID, the latter of which was dominated by successive losses of water. Additionally, the fragments obtained with CTD were more structurally informative and indicative of the radical fragmentation produced in high energy techniques like EID and XUV-DPI by others. CTD of singly protonated Nylon-6,6 produced an abundant CTnoD oxidation peak with a charge of 2+, but no fragments. CID of the isolated 2+ radical at the MS3 level provided a rich spectrum with excellent coverage of the polymer sequence. In contrast, CID generated only successive monomer losses and water losses.

For leucine and isoleucine differentiation, model peptides and wild-type peptides containing leucine or isoleucine were fragmented in their 1+ and 2+ charge states with CTD. The results are compared to CID. The four model peptides of RGGGGXXGGGGR, where X is either Leu or Ile, were indistinguishable with CID, but CTD distinguished the four isomers by providing diagnostic side chain cleavages of the type d- and w ions. These d- and w ions were also apparent in CTD fragmentation of the wild-type peptides, which enabled the correct identification of the Leu and Ile residues in the wild-type peptides.

For the determination of amino acid epimers using CTD, experiments focused on differentiating four isomers of aspartic acid—L-Asp, L-isoAsp, D-Asp, and D-isoAsp—within linear peptides. Wild-type peptides, including the 1+ and 2+ charge states of FVIFLDVK, GYQYLLEPGDFR, and HFSPEDLTVK, were also subjected to CTD, and the results were compared with CID of the same precursors. CTD produced characteristic fragment ions at c+57 for isoAsp residues, but not for Asp residues. Thus, the two isomers can be readily distinguished based on these fragments. These results are supported by the observations of others using electron capture dissociation (ECD) and electron transfer dissociation (ETD). Additionally, the relative intensities of z ions are found to be useful in identifying isoAsp residues. For epimers of Asp, CTD delivers a degree of chiral differentiation similar to or greater than radical directed dissociation (RDD), and a characteristic ion with the identity bn-45 Da or an-NH3 ion can be used to aid in the identification of D-epimers of Asp and isoAsp. For example, R values of 41.0 and 37.5 were produced with CTD of HFSPEDiLTVK at the 1+ and 2+ charge state, respectively. For CID of the same peptide, the R values were only 7.9 for the 1+ charge state and 2.8 for the 2+ charge state.

In summary, CTD provides structurally informative fragmentation of biomolecules that is quite similar to other high-energy fragmentation techniques like ECD, HECD, RDD, and XUV-DPI. CTD has the added benefit of being implemented on a benchtop instrument, allowing for greater accessibility. Furthermore, CTD can be applied to low charge state precursors, which permits its application in studies of singly charged molecules that are unreachable with electron-based fragmentation methods. These advantages afforded to CTD position it to be an invaluable tool in the study of biomolecules.

Embargo Reason

Publication Pending

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