Volume 148, 15 June 2018, Pages 213–222

Neurotoxicity fingerprinting of venoms using on-line microfluidic AChBP profiling

  • a AIMMS Division of BioMolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands
  • b Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands
  • c Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St Lucia, Brisbane, Queensland, 4072, Australia
  • d Department of Biological Sciences, Faculty of Science, National University of Singapore, 16 Science Drive 4, 117558, Singapore
  • e Department of Biological Sciences, Stetson University, 421 N. Woodland Blvd, Unit 8264, DeLand, FL, 32723, USA
  • f hyphen MassSpec, Margrietstraat 34, 2215 HJ, Voorhout, The Netherlands
  • g Alistair Reid Venom Research Unit, Parasitology Department, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK
  • h Research Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK


A high throughput in vitro screening strategy for estimating severity and elements of neurotoxicity caused by snake venoms.

The microfluidic High Resolution Screening system rapidly generates neurotoxicity fingerprints of venoms.

A correlation between neurotoxicity in patients and AChBP affinity profiles measured is demonstrated with 47 snake venoms.

The workflow can be extended to include full structural identification and biological assessment of bioactive venom toxins.


Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins.


  • Microfluidic HRS;
  • Neurotoxicity fingerprinting;
  • AChBP;
  • α7-nAChR;
  • On-line bioaffinity;
  • Nanolc-MS;
  • Elapid venom profiling

1. Introduction

Many studies investigating the toxicity of individual venom components involve the initial activity screen of crude venoms followed by fractionation and re-screening of the fractions. Active fractions for the toxic effect under study are then structurally characterized, mainly by mass spectrometry (MS) based approaches. To characterize the full biological effect, relatively large quantities of toxins are needed, which are acquired by semi-preparative purification, by production in recombinant protein production systems, or by peptide synthesis. The purified bioactives can also be used to study their effect on pharmaceutically relevant receptors or enzymes in venom-based drug discovery, which is a growing research field providing new drug-leads from venoms as natural sources (Casewell et al., 2013; Kini and Fox, 2013; Vetter et al., 2011 ;  Vonk et al., 2011).