Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms.

Autor: Otvos RA; AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. r.a.otvos@vu.nl.; Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. r.a.otvos@vu.nl., Iyer JK; Department of Biological Sciences, Faculty of Science, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore. janaki@u.nus.edu., van Elk R; Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands., Ulens C; Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, Herestraat 49, PB 601, B-3000 Leuven, Belgium. chris.ulens@med.kuleuven.be., Niessen WM; AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. w.m.a.niessen@vu.nl.; Hyphen MassSpec, de Wetstraat 8, 2332 XT Leiden, The Netherlands. w.m.a.niessen@vu.nl., Somsen GW; AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. g.w.somsen@vu.nl., Kini RM; Department of Biological Sciences, Faculty of Science, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore., Smit AB; Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. guus.smit@vu.nl., Kool J; AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. j.kool@vu.nl.
Jazyk: angličtina
Zdroj: Toxins [Toxins (Basel)] 2015 Jun 24; Vol. 7 (7), pp. 2336-53. Date of Electronic Publication: 2015 Jun 24.
DOI: 10.3390/toxins7072336
Abstrakt: The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach.
Databáze: MEDLINE
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