Spectroscopic studies of peptide fragments produced by collision-induced dissociation

Tandem mass spectrometry (MS/MS) combined with collision-induced dissociation (CID) is a key method for primary structure determination of peptides and proteins. Collisionally activated peptides undergo statistical dissociation, forming a series of backbone bn and yn fragment ions that reflect the peptide sequence. However, nascent CID fragments may undergo sequence permutation via "head-to-tail" cyclization, and upon further dissociation give rise to non-direct fragments, hindering the mass spectral interpretation. In this thesis work laser spectroscopy in a cold ion trap is used to provide structural information on the nascent fragments, which can help elucidate the fragmentation mechanisms and predict the occurrence of sequence permutation. The first part of the work is devoted to the b6 fragment ion formed from the protonated model peptide [FAGFAGPG + H]+. For this species five unique conformations have been identified under our experimental conditions. Nitrogen-15 isotopic substitution of individual amino acid residues reveals two intriguing totally symmetric cyclic species. This analysis also provides unambiguous evidence for proton migration during CID for the remaining asymmetric conformers. This information, as well as the vibrational band assignments, simplified the conformational search performed by simulated annealing combined with DFT calculations. The superb agreement between experiment and theory provided a high degree of confidence in the determined structures. To our knowledge this is the first conformer-selective spectroscopic identification of the cyclic bn ions implicated in sequence scrambling during CID. The second part describes the experiments in which the barriers to conformational isomerization have been probed by IR hole-filling spectroscopy (HFS). The HFS recorded in the fundamental as well as in the first overtone NH stretch vibration regions are presented. The next part describes the experiments in which the influence of amino acid composition and length of bn ions on their structural aspects have been investigated. The b6 ions from [FAVFAGPG + H]+ and [YAGYAGPG + H]+ are described first. Based on the comparison of their IR-UV spectra with those of b6 [FAGFAGPG + H]+, some structural features of the former are deduced. For the larger b8 ion from [FAVGFAVGPG + H]+, we have identified six unique conformers, one of which has a totally symmetric cyclic structure. The last part describes construction and characterization of a new cryogenic octopole ion trap. Its advantages over the previously used 22-pole trap are demonstrated.

Rizzo, Thomas
Lausanne, EPFL
Other identifiers:
urn: urn:nbn:ch:bel-epfl-thesis6387-7

Note: The status of this file is: EPFL only

 Record created 2014-10-01, last modified 2018-03-17

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