Venom On-A-Chip: a Fast and Efficient Method for Comparative Venomics
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toxins Article Venom On-a-Chip: A Fast and Efficient Method for Comparative Venomics Giulia Zancolli 1,*, Libia Sanz 2, Juan J. Calvete 2 and Wolfgang Wüster 1,* 1 Molecular Ecology and Fisheries Genetics Lab, School of Biological Sciences, Bangor University, Bangor LL57 2UW, UK 2 Venomics and Structural Proteomics Laboratory, Instituto de Biomedicina de Valencia, CSIC, Jaume Roig 11, Valencia 46010, Spain; [email protected] (L.S.); [email protected] (J.J.C.) * Correspondence: [email protected] (G.Z.); [email protected] (W.W.) Academic Editor: Kevin Arbuckle Received: 20 April 2017; Accepted: 24 May 2017; Published: 28 May 2017 Abstract: Venom research has attracted an increasing interest in disparate fields, from drug development and pharmacology, to evolutionary biology and ecology, and rational antivenom production. Advances in “-omics” technologies have allowed the characterization of an increasing number of animal venoms, but the methodology currently available is suboptimal for large-scale comparisons of venom profiles. Here, we describe a fast, reproducible and semi-automated protocol for investigating snake venom variability, especially at the intraspecific level, using the Agilent Bioanalyzer on-chip technology. Our protocol generated a phenotype matrix which can be used for robust statistical analysis and correlations of venom variation with ecological correlates, or other extrinsic factors. We also demonstrate the ease and utility of combining on-chip technology with previously fractionated venoms for detection of specific individual toxin proteins. Our study describes a novel strategy for rapid venom discrimination and analysis of compositional variation at multiple taxonomic levels, allowing researchers to tackle evolutionary questions and unveiling the drivers of the incredible biodiversity of venoms. Keywords: Bioanalyzer; Crotalus; on-chip electrophoresis; population-level variation; venom composition 1. Introduction Animal venoms are highly complex cocktails of bioactive toxin peptides and proteins, tailored by natural selection to act on specific molecular targets in order to subdue prey or deter predators [1]. By virtue of their high specificity and potency, animal toxins have also been exploited as models for drug development and biodiscovery [2–4], structural biology [5,6], as well as in studies of molecular evolution [7–9]. Although the potential medical value of venom toxins has been contemplated since ancient times, large-scale in-depth characterization of venoms of diverse origins has only become feasible in the last decades. This is especially thanks to the unprecedented advances in “-omics” technologies, which have allowed the recognition of a new field of research, “venomics” [10]. This has led to the establishment of a standard approach for the characterization of venom proteomes [11,12]: briefly, crude venom is fractionated by reverse-phase HPLC (RP-HPLC), followed by SDS-PAGE of the RP-HPLC protein fractions, determination of molecular masses by mass spectrometry, in-gel tryptic digestion of protein bands, and internal amino acid sequence determination by nanospray-ionization CID-MS/MS of selected tryptic peptide ions. This bottom-up peptide-centric pipeline is the most widely used for qualitative and quantitative proteomic analyses of venom components from medically important snake species (see Table1 in Calvete 2013) [ 13], but it has also been employed for comparative studies aimed at investigating patterns of venom composition between different species [14–16], as well as between distinct populations of the same species [17–19]. Toxins 2017, 9, 179; doi:10.3390/toxins9060179 www.mdpi.com/journal/toxins Toxins 2017, 9, 179 2 of 15 Toxinscomposition2017, 9, 179 between different species [14–16], as well as between distinct populations of the 2same of 15 species [17–19]. 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Because adaptation of these and characteristics, prey coevolution venom [21–25]. represents However, an idealinvestigating model system patterns for of understanding population‐level ecological variation adaptation requires and large prey sample coevolution sizes to [detect21–25]. potentially However, investigatingsubtle patterns patterns and correlations of population-level with statistical variation confidence requires [21]. large Thus, sample a method sizes to that detect allows potentially rapid, subtledetailed, patterns consistent and and correlations repeatable with characterization statistical confidence of multiple [21 venom]. Thus, samples a method is needed. that allows At present, rapid, detailed,RP‐HPLC consistent chromatograms and repeatable are commonly characterization used for of this multiple purpose. venom For samples instance, is needed. Saviola Atet present,al. 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Overlapping of reverse-phase HPLC chromatograms of Crotalus scutulatus crude venom from Figure 1. Overlapping of reverse‐phase HPLC chromatograms of Crotalus scutulatus crude venom the same individual sample, highlighting differences between independent runs. The same protein from the same individual sample, highlighting differences between independent runs. The same fractions can have different elution times (A); peak shapes (B); or intensities (C) between runs, resulting protein fractions can have different elution times (A); peak shapes (B); or intensities (C) between runs, in difficult and unreliable scoring. resulting in difficult and unreliable scoring. AsAs anan alternativealternative approach, approach, Gibbs Gibbs & & Chiucchi Chiucchi [17 [17]] analyzed analyzed population-level population‐level venom venom variation variation in thein the massasauga, massasauga,Sistrurus Sistrurus catenatus catenatus, by means, by ofmeans comparisons of comparisons of band presence/absence of band presence/absence on Coomassie on blue-stainedCoomassie blue SDS-PAGE.‐stained However,SDS‐PAGE. SDS-PAGE However, is aSDS laborious‐PAGE method, is a laborious and gel method, image analysis and gel requires image specificanalysis software, requires specific which issoftware, often not which freely is available,often not freely in order available, to score in bandsorder to in score an objective bands in and an standardizedobjective and way.standardized way. 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