Hydrogen / Deuterium Exchange and Fragmentation of Biomolecules to Probe Gas Phase Structure and Energetics

by Herrmann, Kristin Ann.

Presented in this dissertation are FT-ICR H/D exchange and fragmentation studies of protonated peptides for the purpose of better understanding gas phase conformation and protonation motifs, and their affect on fragmentation patterns. In addition, a new ion activation method is developed to enhance the abundance of higher energy fragmentation pathways, thereby providing additional structural and/or mechanistic information. Studies to probe the relay mechanism of H/D exchange in small, model peptides found that residue position, proline configuration, and availability of the termini have a measurable effect on the H/D exchange behavior reflective of different conformation / protonation motifs. Charge remote cleavage C-terminal to aspartic acid was studied with the fixed charge derivative tris(2,4,6-trimethoxyphenyl) phosphonium (tTMP-P+). Ab initio calculations demonstrate that tTMP-P+ cannot activate the proposed aspartic acid nucleophile, the acidic side chain hydrogen initiates cleavage. Despite the absence of an ionizing proton, the derivatized peptide P+LDIFSDF (where P+ = tTMP-P+) exchanges three acidic hydrogens for deuterium. This supports the proposed aspartic acid cleavage mechanism that occurs without the direct involvement of an ionizing proton because acidic protons are able to participate in H/D exchange in the absence of an added proton. In addition, H/D exchange of P+LDIFSDF analogues provides insight into the sites and mechanisms of H/D exchange. 21 H/D exchange to separate distinct peptide populations with differing rates and their subsequent fragmentation demonstrate that conformation / protonation motif affects the fragmentation spectrum observed. Studies of [P+LDIFSDF + H]2+, [RPPGFSPFR + 2H]2+ (bradykinin), and [RVYIFPF + 2H]2+ show that at least two distinct structures exist with different rates of H/D exchange and different fragmentation patterns. The overall MS/MS spectrum is a linear combination of all conformations and protonation motifs. In addition, fragmentation of labeled populations suggests that complementary bn+ / ym-n+ ion pairs are being formed from doubly-charged precursor by the same mechanism. SORI-RE CID is a new FT-ICR ion activation method based on the combination of sustained off-resonance irradiation (SORI) and on-resonant excitation (RE) with the purpose of enhancing higher energy fragmentation while maintaining low energy processes. The experiments presented serve to illustrate the usefulness of SORI-RE in diverse cases. 22