Structural and conformational aspects of gas phase peptides probed by electron capture dissociation

Abstract

Mass spectrometry (MS) is a major player in the field of structural characterization of peptides and proteins. MS-based proteomics is nowadays routinely carried out at universities, research institutes and hospitals. Often, fragmentation techniques are applied to generate information about the molecular architecture. One way to obtain peptide fragments is by making use of reactions between multiply charged polypeptide cations in the gas-phase and low energy electrons. This process is called electron capture dissociation (ECD) and leads to high sequence coverage without affecting non-covalent interactions. The properties of ECD enable both identification of polypeptides and investigations on protein folding processes and protein-complexes. It is of fundamental importance to understand the underlying mechanism of ECD. Thus resulting knowledge opens new possibilities for further method development. In this thesis, a fundamental study on ECD is described, as well as instrumental development and applications to biologically relevant biomolecules. All experiments were carried out using a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). To asses the role of conformational heterogeneity of gas-phase peptides we performed ECD on two peptides, one linear and one cyclic, at extreme low temperatures. It was found that at 85 K, ECD yields for both peptides a lower number of backbone fragments compared to ECD at room temperature under identical conditions. It was concluded that extensive sequence coverage in ECD can be obtained by increasing the conformational heterogeneity of the precursor ion population. Based on a novel approach, an instrumental setup was developed to increase the conformational heterogeneity of the ion cloud by heating it with infrared photons. This allows sequential or simultaneous on-axis infrared multiphoton dissociation (IRMPD) and ECD in the ion cyclotron resonance (ICR) cell. As a result, the informational content of the MS/MS spectra has improved at time scales compatible with chromatographic separation techniques.

High sequence coverage is difficult to obtain for peptides and proteins that contain monosulfide- or disulfide bridges using fragmentation methods based on vibrational excitation. In this work it is shown that complexation of such polypeptides with divalent metal cations resulted in very rich sustained off-resonance - collision induced dissociation (SORI-CID) mass spectra. Under these circumstances the disulfide bond is cleaved up to 50% of the total product ion intensity. We suggest that the observed disulfide bond cleavage for peptide-metal complexes reflects conformational changes induced by the presence of the metal ion. Moreover, the metal binding site can be estimated based on complementary fragments from peptide-metal complexes. ECD applied to these complexes displays not only differences but also similarities. Such common features can be indicative for structural similarities between different species.

The results presented here contribute to a better understanding and further development of ECD as a useful fragmentation technique. Future proteomics studies will greatly benefit from ECD due to its ability to produce extensive sequence coverage and information about conformation and subtle structural changes in peptides and proteins.