Mass spectrometry of biologically active carbohydrates and their interactions with proteins
Dr. Michael Mormann
PhD projects to be supervised by the members of the Biomedical Mass Spectrometry group should be designed for the characterization of non-covalent interaction of carbohydrate-binding proteins (CBPs) with free glycans and glycoconjugates acting as specific binding ligands by use of mass spectrometric techniques and related methodologies. The MS-based investigation of non-covalent complexes from both condensed phase and gas phase will yield a more profound insight into the nature of carbohydrate-protein interactions.
Taking advantage of instrument specifications, a comprehensive structural analysis of both host and ligand molecules is feasible. CBPs such as lectins are characterised with respect to their amino acid sequence, glycosylation sites and glycan structure by means of various techniques already established in our laboratory. MS methodologies are employed to obtain both structural and thermochemical parameters of non-covalent protein-sugar complexes from the condensed and gas phase. Electron Capture Dissociation experiments will aim at mapping of the interfacial region between ligand and host. Thermodynamic data such as equilibrium association constants are evaluated using various direct ESI-based binding assays. Control experiments employing real-time biomolecular interaction analysis (e.g. surface acoustic wave, SAW) are performed to validate data obtained from MS experiments. Projects should be designed to be performed in the Biomedical Mass Spectrometry lab at the University of Münster, but a part of each proposed project will be carried out in collaborating labs in India. Following tentative titles are proposed for PhD theses:
1. Unravelling the binding topology of non-covalent carbohydrate-protein complexes
Structural parameters on non-covalent protein-sugar complexes from the condensed and the gas phase will be obtained by MS techniques. Mapping of the interfacial region between ligand and host is performed by hydrogen/deuterium exchange and elelctron capture dissociation (ECD) experiments (Collaborations will be with Moerschbacher, Siva Kumar, and Swamy).
2. Probing the thermodynamics and kinetics of non-covalent lectin-carbohydrates complexes
This project comprises the isolation and characterisation of either a plant or invertebrate lectin in collaboration with Swamy and Siva Kumar followed by determination of binding specificity. Equilibrium constants and kinetics of non-covalent protein-carbohydrate complex formation will be measured by MS-based techniques, ITC measurements (in collaboration with Swamy), and SAW experiments (collaboration with Müthing).
3. Understanding inhibition of matrix metalloproteases (MMP) by chitosan oligomers
This project encompasses the structural characterization of degree of polymerisation (DP), the degree of acetylation (DA), and the pattern of acetylation (PA) of chitosan oligomers exhibiting elevated affinity towards MMPs by mass spectrometric techniques. Subsequently, binding thermodynamics and kinetics will be unravelled by complementary MS and SAW experiments (with Moerschbacher, Müthing; synthesis of standards together with Balamurugan, Ramu Sridhar)
4. Development of a mass spectrometry-based platform for glycosaminoglycan (GAG) quantitation
In collaboration with Götte, Grobe and Seidler a method for GAG quantitation based on TLC separation of fluorochrome-derivatised GAG analytes followed by direct MS analysis will be established.
Beside the proposed topics, additional ones of similar type can be proposed by the candidates and are welcome!
