Advances in the mass spectrometry-based analysis of biomolecules and their covalent modifications: a molecular window on some therapeutic approaches

Date: 25 November 2016

Venue: Universiteit Antwerpen, Campus Groenenborger, U0.25 - Groenenborgerlaan 171 - 2020 Antwerpen (route: UAntwerpen, Campus Groenenborger)

Time: 3:30 PM

Organization / co-organization: Department of Chemistry

PhD candidate: Debbie Dewaele

Principal investigator: Filip Lemière

Short description: PhD defence Debbie Dewaele - Faculty of Science - Department of Chemistry


With an estimated death rate of 12 million by the year 2020 cancer is one of the leading causes of death, therefore the optimisation of current cancer therapies (e.g. chemotherapy) and the development of new perhaps less invasive cancer treatments (e.g. aptamer based treatments) is absolutely desirable. Analytical techniques have proven to be powerful in this studies, especially mass spectrometry has evolved to one of the most sophisticated and diverse analytical methods, providing several approaches that are able to contribute to this research. This PhD thesis discusses the use of different mass spectrometry-based approaches in the scope of three different cancer treatments.

In a first chapter, using a wide range of mass spectrometry-based methods, I have studied the formation of covalent drug adducts on proteins. As a model system, the nitrogen mustard derivate melphalan was used to study the adduct formation. I determined the reactivity of melphalan towards the different free amino acids, resulting in the observation of adduct formation. In summary, adduct formation was observed for the following amino acid side chains: the thiol group of cysteine and the thioether group of methionine, the phenolic group of tyrosine, the carboxylic side chains of aspartic and glutamic acid, ε-amino group van lysine and the imidazole moiety of histidine. Furthermore, the data confirmed melphalan alkylation of the carboxylic acid and amino groups of amino acids, which are models for the C- and N- termini in peptides or proteins. Next to it, the different alkylation products show distinctive fragmentation patterns which enable, in many but not in all cases, a fast identification of the different melphalan adducts (i.e. cysteine and lysine). For the protein-melphalan adducts, I determined the extent of melphalan-alkylation using a combined methodological approach of bottom-up CID and top-down ETD. Using this approach we were able to pinpoint the melphalan alkylation to a number of amino acid residues in the protein. Mapping the identified modification sites on the 3D structure of the proteins showed that the adduct formation is not solely explained by the nucleophilicity of the amino acids in a protein sequence, but also depends on the accessibility and/or flexibility of the residues. Ion mobility analysis of the two model proteins incubated with melphalan however did not reveal any discernible effect on the cross section, i.e. 3D structure, of the proteins.

The effect of oxygen plasma treatment on short oligosaccharides and proteins is discussed in the second part. I showed that that oxygen plasma treatment has a big effect on the stability and integrity of short oligosaccharides, resulting in glycosidic bond cleavage. These first results could point to a possible explanation for the antibacterial effect of plasma treatment on biofilms. The oxygen plasma treatment could be affecting the oligosaccharides within the EPS of the biofilms, resulting in a lower resistance, but additionally it could also affect the integrity of the cell wall with cell death as an outcome. Next to it, I determined the effect of ROS on peptides and proteins. I showed that, just like the melphalan adduct formation, the formation of oxidation products is not solely dependent on the susceptibility of the amino acid residues but is also strongly influenced by the accessibility of the amino acid residues in the proteins. This thesis furthermore shows that mapping the modifications sites (i.e. melphalan-alkylation sites and oxidation sites) on the 3D structure of proteins is extremely helpful in obtaining a better understanding of the precise mode of action.

In the final part of this thesis, we demonstrated the additional value of mass spectrometry in the study of DNA aptamers and their non-covalent ligand complexes. We have evaluated the use of ion mobility in the context of aptamers and their ligand complexes and used the FWHM of the drift time profiles to study the flexibility of the aptamers and their complexes. Furthermore, combining linear ion mobility experiment together with CIU experiments we proposed the peripheral binding model as a possible binding model for aptamer quinine complex formation.