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Enzymes are often considered less 'druggable' targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), [...]

A cold-cathode electron impact ionization source based on field emission from an array of diamond-coated silicon whiskers is described. The source is coupled to a microscale ion trap mass spectrometer (r(sub 0) = 0.50 mm, z(sub 0) = 0.50 mm). An electron beam of 250 nA could be obtained through the 0.45-mm diameter opening in the end cap electrode.

Two-laser ion trap mass spectrometry and X-ray photoelectron spectroscopy were used to investigate the chemical modification of polystyrene thin films by 10-100 eV SF5+, C3F5+ and SO3 ions. Results showed that different mechanisms of fluorination were induced on the polymer surface by SF5+ and C3F5+ ions. Moreover, different aromatic and aliphatic fluorine bonding fields were present on both SF5+ and C3F5+ modified thin films.

Several improvements were made on the two-laser ion trap mass spectrometry (L2ITMS) device used for organic surface analysis. First, a vacuum ultraviolet radiation used for photoionization is installed. Second, the desorption and ionization regions are repositioned and the photoionization laser beam is positioned off-center of the ring electrode. These improvements allow tandem mass spectrometry of low-concentration surface species and increases the number of species that can be ionized.

This paper describes two-laser ion trap mass spectrometry (L2ITMS) for the analysis of organic, polymeric, and biological surfaces. L21TMS uses one laser to desorb intact molecules from a surface, a second laser for photoionization, and an ion trap for single or tandem mass spectrometric analysis. We demonstrate the capabilities of this instrument by performing laser desorption/photoionization/tandem mass spectrometric analyses of amino acid and mixed polymer thin films. We then use L2ITMS in conjunction with X-ray photoelectron spectroscopy to chemically analyze the surface of an ion-bombarded polystyrene thin film. Low-energy S[[F.sub.5].sup.+] ion bombardment causes fluorination of intact polystyrene, partial destruction of the aromaticity of the polystyrene, Fluorination of the resultant nonaromatic organic material, and sputtering of the polystyrene. The first of these dominates at 50 eV ion energy, while the latter three dominate at 250 eV.

3',5'-Cyclic adenosine monophosphate (cAMP) is a common intracellular second messenger that enables cells to respond to external stimuli. Measurement of intracellular cAMP concentrations is thus widely used for studying guanosine triphosphate binding protein-coupled receptors (GPCRs), which make up a large class of pharmaceutical drug targets. Although several assay technologies exist to measure cAMP, most are not suitable for ultra-high-throughput screening (uHTS), as is often required for screening large (greater than 1 million) chemical libraries for the identification of suitable leads for drug development. Here we report that the enzyme fragment complementation assay, a homogeneous gain of signal assay based on complementation of two fragments of a beta-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-microl total assay volume in 3,456-well plate format. We describe the miniaturization of this assay into 3,456-well plate format exhibiting comparable sensitivity and plate statistics to those of a 384-well assay and the application of this assay in uHTS for the identification of antagonists of a Gi-coupled receptor.

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.