| This dissertation describes the development of a new method for monitoring protein-ligand interactions using a Lab-on-Valve apparatus coupled to UV/visible and electrospray ionization mass spectrometry (ESI-MS). The system provides fast regeneration of assay surfaces, monitors binding/dissociation of analytes in situ, and allows screening of compound mixtures. It is capable of providing kinetic information as well as equilibrium dissociation and association constants. In addition, it is also useful as a tool for purification and on-line ESI-MS analysis of biotin-containing conjugates. The liquid/bead handling is fully automated, precise, and robust.; The system was used to determine the dissociation rate constants of two chromophore-tagged biotin conjugates for release from immobilized streptavidin. The study revealed that the use of an N-methylalanine (sarcosine) linker in the conjugate and methanol in the solvent system dramatically increased the dissociation rates. Following these findings, affinity capture and release processes of biotin-containing substrate and product conjugates on immobilized streptavidin were automated for several enzyme activity assays. Previously, the elution step required at least 1 hour to retrieve a sufficient amount of samples for MS quantification, in contrast, with LOV-ESI-MS apparatus, the entire purification and MS analysis steps were automated and executed within 5 minutes. Fast analysis and robust operation (60 flawless repetitive analyses) made this technology promising for transfer into clinical practice.; The LOV-ESI-MS apparatus was also used to determine the equilibrium dissociation constants of compound mixtures against immobilized cholera toxin (B subunit) and T. brucei Pex5 receptor protein. In addition, compound mixtures were screened against human Pex5 receptor protein and 5 other proteins relevant to pathogenic protozoa, in which the relative affinity of each analyte in the mixtures was determined. With an addition of an autosampler, the system would be used for unattended analysis for routine screening necessary in various stages of drug development. |
