title: Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.
abstract: 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.
authors.affiliation: FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, New Jersey, USA. tom_holt@mac.com
authors.forename: Tom G
authors.initials: TG
authors.lastname: Holt
authors.forename: Bernard K
authors.initials: BK
authors.lastname: Choi
authors.forename: Neil S
authors.initials: NS
authors.lastname: Geoghagen
authors.forename: Kristian K
authors.initials: KK
authors.lastname: Jensen
authors.forename: Qi
authors.initials: Q
authors.lastname: Luo
authors.forename: William A
authors.initials: WA
authors.lastname: LaMarr
authors.forename: Gergely M
authors.initials: GM
authors.lastname: Makara
authors.forename: Lorraine
authors.initials: L
authors.lastname: Malkowitz
authors.forename: Can C
authors.initials: CC
authors.lastname: Ozbal
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Xiong
authors.forename: Claude
authors.initials: C
authors.lastname: Dufresne
authors.forename: Ming-Juan
authors.initials: MJ
authors.lastname: Luo
mesh_terms: D000818:Animals; D002138:Calibration; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003540:Cystathionine / Q000032:analysis*; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D007202:Indicators and Reagents; D003540:Cystathionine