Label-Free High-Throughput Screening via Mass Spectrometry: A Single Cystathionine Quantitative Method for Multiple Applications
| Autor: | Kristian K. Jensen, Qi Luo, Ming-juan Luo, Neil S. Geoghagen, Yusheng Xiong, Gergely M. Makara, Claude Dufresne, William A. LaMarr, Bernard K. Choi, Lorraine Malkowitz, Can C. Ozbal, Tom G. Holt |
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| Rok vydání: | 2009 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization High-throughput screening Drug Evaluation Preclinical Mass spectrometry Mass Spectrometry Cell Line Mice chemistry.chemical_compound Cystathionine Drug Discovery Animals Humans Derivatization Chromatography High Pressure Liquid Chromatography Dose-Response Relationship Drug biology Drug discovery Multiple applications Cystathionine beta synthase Enzyme assay Mice Inbred C57BL Kinetics chemistry Linear range Data Interpretation Statistical Calibration biology.protein Molecular Medicine Indicators and Reagents |
| Zdroj: | ASSAY and Drug Development Technologies. 7:495-506 |
| ISSN: | 1557-8127 1540-658X |
| DOI: | 10.1089/adt.2009.0200 |
| Popis: | Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project. |
| Databáze: | OpenAIRE |
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