title: Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry.
abstract: This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC-FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist-agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist-agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup.
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.|Department of Molecular and Cellular Neurobiology, Vrije Universiteit Amsterdam , De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Reka A
authors.initials: RA
authors.lastname: Otvos
authors.affiliation: Department of Molecular and Cellular Neurobiology, Vrije Universiteit Amsterdam , De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Pim
authors.initials: P
authors.lastname: van Nierop
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.|hyphen MassSpec, Herenweg 95, 2361 EK Warmond, The Netherlands.
authors.forename: Wilfried M A
authors.initials: WM
authors.lastname: Niessen
authors.affiliation: Department of Biological Sciences, National University of Singapore , 14 Science Drive 4, 117543, Singapore.
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Department of Molecular and Cellular Neurobiology, Vrije Universiteit Amsterdam , De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002853:Chromatography, Liquid; D005434:Flow Cytometry; D006801:Humans; D013058:Mass Spectrometry; D009862:Online Systems; D014407:Tumor Cells, Cultured