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Biology 2014, 3(1), 139-156; doi:10.3390/biology3010139
Article

Miniaturized Bioaffinity Assessment Coupled to Mass Spectrometry for Guided Purification of Bioactives from Toad and Cone Snail

1,†
, 1,2,†
, 3
, 2
, 1
, 4
, 5
, 5
, 3
, 2
, 1,6
 and 1,*
1 AIMMS Division of BioMolecular Analysis, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1083 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 Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands 4 AIMMS Division of Organic Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1083 HV Amsterdam, The Netherlands 5 The Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia 6 MassSpec Consultancy, de Wetstraat 8, 2332 XT Leiden, The Netherlands These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 9 December 2013 / Revised: 23 January 2014 / Accepted: 26 January 2014 / Published: 13 February 2014
(This article belongs to the Special Issue Screening for Biologically Active Compounds)
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Abstract

A nano-flow high-resolution screening platform, featuring a parallel chip-based microfluidic bioassay and mass spectrometry coupled to nano-liquid chromatography, was applied to screen animal venoms for nicotinic acetylcholine receptor like (nAChR) affinity by using the acetylcholine binding protein, a mimic of the nAChR. The potential of this microfluidic platform is demonstrated by profiling the Conus textile venom proteome, consisting of over 1,000 peptides. Within one analysis (<90 min, 500 ng venom injected), ligands are detected and identified. To show applicability for non-peptides, small molecular ligands such as steroidal ligands were identified in skin secretions from two toad species (Bufo alvarius and Bufo marinus). Bioactives from the toad samples were subsequently isolated by MS-guided fractionation. The fractions analyzed by NMR and a radioligand binding assay with α7-nAChR confirmed the identity and bioactivity of several new ligands.
Keywords: on-line microfluidics; nano-liquid chromatography-mass spectrometry (nano-LC-MS); Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP); Conus textile; Bufo alvarius; Bufo marinus on-line microfluidics; nano-liquid chromatography-mass spectrometry (nano-LC-MS); Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP); Conus textile; Bufo alvarius; Bufo marinus
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.

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MDPI and ACS Style

Heus, F.; Otvos, R.A.; Aspers, R.L.E.G.; van Elk, R.; Halff, J.I.; Ehlers, A.W.; Dutertre, S.; Lewis, R.J.; Wijmenga, S.; Smit, A.B.; Niessen, W.M.A.; Kool, J. Miniaturized Bioaffinity Assessment Coupled to Mass Spectrometry for Guided Purification of Bioactives from Toad and Cone Snail. Biology 2014, 3, 139-156.

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