RESUMO
A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFiretrade mark platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z' value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target.
Assuntos
Carboxiliases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Espectrometria de Massas/métodos , Carboxiliases/análise , Carboxiliases/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Congelamento , Humanos , Rim/citologia , Cinética , Plasmídeos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Robótica , Análise de Sequência de DNA , TransfecçãoRESUMO
An IgG mouse monoclonal antibody (10F05) against polyethylene glycol has been generated. The antibody reacts with PEG regardless of the linker used for PEG attachment, and is able to recognize a PEGylated peptide in plasma at concentrations as low as 3 pg/mL. The antibody is readily purified in substantial quantities. The PEG IgG will find significant utility in the sensitive detection of PEG derivitives during the pharmacokinetic characterization of PEGylated compounds.
Assuntos
Anticorpos Monoclonais/biossíntese , Polietilenoglicóis/química , Animais , Antígenos/química , Ensaio de Imunoadsorção Enzimática , Hemocianinas/imunologia , Hibridomas/imunologia , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologiaRESUMO
A new method to measure the activity of lipid-metabolizing enzymes is described. Subsequent to an enzymatic reaction, a two-phase system (organic/aqueous) is established by the addition of a phase partition scintillation fluid (PPSF). The PPSF serves as a scintillation fluid, a phase partition agent, and a carrier/separator of an organic-soluble radiolabeled reaction substrate or product. Applying an empirically derived set of conditions typically enhances the separation of substrate from product whereby one species is effectively solubilized in the PPSF. In situ partitioning of the radionuclide-containing organic/lipid phase from the aqueous phase occurs within individual wells of 96-well or 384-well density PPSF-resistant microtiter plates without the requirement for multiple organic solvent extractions and aspirations, making this method applicable to high-throughput screening. The utility of this method for both kinetic characterization and high-throughput screening is demonstrated with acetyl-CoA carboxylase and fatty acid synthase.