title: CE-ESI-MS/MS as a rapid screening tool for the comparison of protein-ligand interactions.
abstract: In drug development, the combinatorial synthesis of drug libraries is common use, therefore efficient tools for the characterization of drug candidates and the extent of interaction between a drug and its target protein is a central question of analytical interest. While biological activity is tested today by enzyme assays, MS techniques attract more and more attention as an alternative for a rapid comparison of drug-target interactions. CE enables the separation of proteins and drug-enzyme complexes preserving their physiological activity in aqueous media. By hyphenating CE with ESI-MS/MS, the binding strength of enzyme inhibitors can be deduced from MS/MS experiments, which selectively release the inhibitor from the drug-target complex after CID. In this study, alpha-chymotrypsin (CT), a serine protease, was chosen as a model compound. Chymostatin is a naturally occurring peptide aldehyde binding to CT through a hemiacetal bond and electrostatic interaction. First, a CE separation was developed, which allows the analysis of alpha-CT and a chymotrypsin-chymostatin complex under MS-compatible conditions. The use of neutral-coated CE capillaries was mandatory to reduce analyte-wall interactions. ESI-quadrupole ion trap-MS was worked out to demonstrate the selective drug release after CID. Fragmentation of the drug-enzyme complex was monitored in dependence from the excitation energy in the ion trap, leading to the V(50) voltage that enables 50% complex fragmentation as a reference value for chymotrypsin-chymostatin complex. A stable CE-ESI-MS/MS setup was established, which preserves the drug-enzyme complexes during ionization-desolvation processes. With this optimized setup, different CT inhibitors could be investigated and compared.
authors.affiliation: Saarland University, Saarbrücken, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hoffmann
authors.affiliation: Saarland University, Saarbrücken, Germany.
authors.forename: Markus M
authors.initials: MM
authors.lastname: Martin
mesh_terms: D002021:Buffers; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D025941:Protein Interaction Mapping / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature
chemical_list: D002021:Buffers; D009842:Oligopeptides; C002101:chymostatin; D002918:Chymotrypsin