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Toxins 2015, 7(7), 2336-2353; doi:10.3390/toxins7072336

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

1
AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
2
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
3
Department of Biological Sciences, Faculty of Science, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
4
Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, Herestraat 49, PB 601, B-3000 Leuven, Belgium
5
Hyphen MassSpec, de Wetstraat 8, 2332 XT Leiden, The Netherlands
*
Author to whom correspondence should be addressed.
Academic Editor: Bryan Grieg Fry
Received: 27 March 2015 / Revised: 6 May 2015 / Accepted: 16 June 2015 / Published: 24 June 2015
(This article belongs to the Special Issue Selected Papers from the 5th Venoms to Drugs Meeting)
View Full-Text   |   Download PDF [1542 KB, uploaded 24 June 2015]   |  

Abstract

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. View Full-Text
Keywords: 5-HT3 receptor; 5HTBP; high-resolution screening; snake venoms; nano-LC-MS 5-HT3 receptor; 5HTBP; high-resolution screening; snake venoms; nano-LC-MS
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Otvos, R.A.; Iyer, J.K.; van Elk, R.; Ulens, C.; Niessen, W.M.A.; Somsen, G.W.; Kini, R.M.; Smit, A.B.; Kool, J. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms. Toxins 2015, 7, 2336-2353.

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