Metodutveckling för karakterisering av ligand-receptor interaktioner in situ med frontal affinitets-kromatografi kopplat till desorptions-elektrospray joniserande masspektrometri

Tidsperiod: 2015-07-01 till 2018-06-30

Projektledare: Erik Jansson

Finansiär: Vetenskapsrådet

Bidragstyp: Bidrag för anställning eller stipendier

Budget: 3 150 000 SEK

The association of seven transmembrane helices with the lipid bilayer of a biological cell is both what structurally defines G-protein coupled receptors (GPCR), and what makes them very difficult to study. Despite advances in genomics, proteomics, and protein structure characterization, there are more than 140 orphan GPCRs which lack the identity of an endogenous ligand. We propose to develop a method where the affinity and kinetics of ligand–receptor interactions can be screened in situ with mass spectrometry (MS). By using desorption electrospray ionization (DESI), our label-free method will be used to study ligand–receptor interactions with high mass and time resolution, where the receptors remain intact under ambient conditions on the cell surface and the ligand of interest is continuously perfused over the cells. We will provide a proof-of concept for our methodology by determination of receptor affinities with known GPCR-ligand models, such as glutamate binding to metabotropic glutamate receptors. Further, we will interface the DESI-MS setup with the capability of optical imaging. By measuring calcium-flux with fluorescent probes in parallel with ligand affinity, our system will distinguish, in realtime, if a binding ligand is an agonist or an antagonist. Finally, we will create peptide libraries in-house from rodent models of nervous and endocrine tissues. Our developed system will screen for interactions between endogenous peptides and orphan GPCRs, potentially discovering novel GPCR-binding peptides. The established techniques and biological results will aid the discovery of new GPCR-binding ligands. Prof. Zare at Stanford University and Prof. Bergquist at Uppsala University will support this project. Their current research focuses on protein-interactions and proteomics using DESI-MS. Their expertise along with state-of-the-art instrumentation will be a valuable asset. In addition to the scientific results achieved, Dr. Jansson will become knowledgeable in cutting edge techniques to further increase national competence of biological MS in Sweden.