High-throughput chemical analysis using capillary electrophoresis and electrospray ionization mass spectrometry with applications to drug screening.

摘要

Ever increasing throughput in chemical analysis is of interest for many endeavors including biochemistry, genetics and pharmacology. One of the most prominent areas is drug discovery where combinatorial libraries are screened for their activities using biochemical assays. Although multi-well plate-based fluorescence assays are highly successful for high-throughput screening, more information rich and label-free assays would be more versatile and offer new opportunities for human therapeutics discovery. In this dissertation, methods for improving throughput of electrophoretic and mass spectrometric assays and their applications to drug screening will be described.;A fluorescent hydrolysis assay was first developed for G protein-coupled receptors (GPCRs). This assay utilized capillary electrophoresis (CE) for fast determination of GPCR activation and proved to be amenable for high-throughput applications. Throughput of assays of this kind was then improved by developing microfluidic chips with up to 36 parallel electrophoresis channels each capable of rapid separation. This system was applied to screening for inhibitors of RGS-G Protein interaction. Further improvement of assay throughput was sought by developing a sample introduction interface which allowed discrete samples in a two-phase flow to be continuously fed to parallel CE separation. The ultimate throughput obtained was no longer limited by the time-consuming sample transferring step but only by speed of CE separation.;In the second approach, mass spectrometry (MS) was explored as an analytical method for label-free enzyme assays. In order to improve the throughput of MS-based assays and fully exploit the fast scanning speed of MS, nanoliter of sample plugs segmented by air were sequentially infused to a conductive nano-electrospray ionization (ESI) emitter tip for fast MS analysis. High-throughput screening (HTS) of acetylcholinesterase inhibitors was demonstrated using this method and a throughput of 0.65 Hz was achieved.