Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases
 (2017) 23:26 
DOI 10.1186/s40409-017-0117-8
REVIEW Open Access
Strategies in ‘snake venomics’ aiming
at an integrative view of compositional,
functional, and immunological
characteristics of venoms
Bruno Lomonte1* and Juan J. Calvete2
Abstract
This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and
discusses how these may be combined through diverse experimental approaches with the goal of achieving a
more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms.
Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere
cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological
(antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary
strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by
venomous snakes, responsible for thousands of deaths every year.
Keywords: Snake venoms, Proteomics, Venomics, Antivenomics, Toxicovenomics
Background the most common snake species of medical relevance,
The potent harmful effects of snake venoms have in- leaving those of species that are scarce, or more difficult to
trigued mankind for centuries, inspiring in many cultures collect and keep captive, largely unexplored.
both fear and fascination [1]. With the advent of modern Following the general trends in biosciences, a new era
science, research on snake venoms has mainly targeted in the characterization of snake venoms began with the
three goals [2–4]: (a) deciphering their biochemical com- introduction of proteomics and related -omics techno-
positions, (b) understanding their mechanisms of action logical tools, which have steered a major and rapid ex-
and potential uses thereof, and (c) devising antidotes for pansion of knowledge on their overall composition.
the treatment of envenomation. Venoms from a growing number of snake species have
Snake venoms are secretions produced by a pair of been, and are being, characterized worldwide by proteomic
specialized exocrine glands, predominantly composed by approaches, providing an unprecedented data platform to
diverse peptides and proteins, many of which are endowed enhance our understanding on these fascinating, but
with enzymatic activities [5, 6]. Most of the current know- dangerous, toxic cocktails. Given that envenomation is
ledge on venoms has been gathered by conventional a relevant cause of morbidity and mortality in the rural
biochemical and pharmacological approaches, where tropics of the world [7, 8], new knowledge on the bio-
particular toxins are first isolated, and then studied in chemical constitution of venoms is of high potential
depth to determine their fundamental structural and impact in medicine, as discussed in the following sections.
mechanistic features. As expected, available information is In addition, omics-based characterization of venoms is
biased towards toxins that are abundant in venoms from unveiling new paths to analyze fundamental questions in
biology [9]. The recruitment of genes and evolution of
* Correspondence: bruno.lomonte@ucr.ac.cr toxic functionalities from ancestral ‘physiological’ protein
1Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa scaffolds, for example, is an area of research largely
Rica, San José 11501, Costa Rica
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 2 of 12
powered by the recent introduction of -omic tech- approaches converge in their goal of obtaining a catalogue,
niques to the study of snake venoms [10–13]. as comprehensive as technically possible, of the protein/
This work offers a general view on the evolving strat- peptide constituents of a given venom, there are differences
egies for the proteomic analysis of snake venoms, and in the overall information that can be obtained, such as the
discusses how these may be combined with diverse ex- possibility of complementing the final qualitative informa-
perimental approaches with the goal of achieving a more tion with an estimation of relative abundances for the
comprehensive knowledge on the compositional, toxic, venom components, or other relevant characteristics. A
and immunological characteristics of venoms. shared limitation of proteomic experiments dealing with
any of the above-mentioned strategies is the paucity of gen-
Proteomic approaches, pro et contra omic/transcriptomic databases for venomous snakes. This
It is commonly said that there is no ‘one-size-fits-all’ situation often restrains the prospect of identifying individ-
among the various analytical strategies available for ex- ual components, leaving only the possibility to assign them
ploring the proteome of complex biological samples, to known protein families on the basis of similarity with
since each approach has its particular advantages and existing sequence entries [19]. Nevertheless, such limitation
disadvantages. Several reviews have previously dealt with has been tackled by performing transcriptomic analyses of
the description of different workflows for proteomic venom glands in combination with the proteomic profiling
characterization of snake venoms [14–18]. Therefore, we of venom [19–22]. This greatly enhances the performance
do not aim to present here a detailed view of their technical of matching algorithms for high-resolution mass spectra
aspects. Rather, we highlight some of the most notable dif- and allows to move from a protein-family resolution, to a
ferences, pro et contra, among them and discuss their po- protein-locus resolution [17]. In addition to the growth of
tential for combination with complementary methods that transcriptomic data, new genomic sequencing data increas-
may expand the informative value of the datasets obtained, ingly reported for venomous snakes [23, 24] will also facili-
in terms of their biological and biomedical significance. tate protein identification by automated mass spectrometry
Snake venom proteomes have been analyzed using essen- (MS) processing software.
tially three decomplexation strategies: (a) two-dimensional
gel electrophoresis (2DE)-based, (b) liquid chromatography Gel-based proteomic strategies
(LC)-based, and (c) combined (LC + 1DE)-based, as Gel-based approaches (Fig. 1a) have been used in several
schematically represented in Fig. 1. While all of these proteomic studies on snake venoms, including some of
Fig. 1 General types of analytical bottom-up strategies employed in the proteomic profiling of snake venoms. a Gel-based strategies involve the
separation of the venom proteins by two-dimensional gel electrophoresis (2DE) followed by staining and spot picking. Protein spots are then
in-gel digested (usually with trypsin, scissors icon) and the resulting proteolytic peptides submitted to tandem mass spectrometry (MS/MS)
analysis. b Liquid-chromatography (LC)-based strategies (shotgun proteomics) digest the whole venom with trypsin and separate the resulting
peptides usually by multidimensional nano-flow HPLC, hyphenated to MS/MS analysis. c The combined strategy of ‘snake venomics’ takes
advantage of the opportunity of performing the fractionation and the quantification of the venom components in the same reversed-phase
chromatography step. A second step of separation and quantification is performed by SDS-PAGE followed by gel densitometry. Protein bands
are excised, in-gel digested with trypsin, and submitted to MS/MS analysis
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 3 of 12
the first reported examples [25–30]. Individual spots are can be obtained by reverse-phase HPLC columns at the
excised, in-gel digested, and submitted to tandem mass nano-flow scale, especially when combined in-line with
spectrometry (MS/MS) analysis. Among advantages, a additional ion-exchange or other types of LC media in so-
full pattern of sample decomplexation can be obtained called ‘2D-LC’ or multidimensional separations. Although
in a single two-dimensional gel electrophoresis (2DE), these strategies are well developed to provide a deep cata-
from which information on the isoelectric point (pI, first loguing of the protein/peptide components of the venom,
dimension) and apparent molecular weight (Mw, second the relationship of the identified peptides to their intact
dimension) of the proteins can be readily determined for parent molecules is essentially lost, or very difficult to re-
each spot. Moreover, the macromolecular organization construct, owing to the fact that digestion is performed on
of venom proteins can also be assessed by comparing the crude venom sample as a whole. Consequently, con-
2DE separations run under non-reducing conditions in version of the obtained qualitative data into a quantitative
both directions versus non-reducing (first dimension)/ estimation of protein abundances becomes complicated.
reducing (second dimension) [31]. Also, it is possible to Current high-end MS instruments and specialized
stain the gel not only for proteins, but also for conjugated software allow for ‘label-free’ (i.e., not depending on the
moieties such as glycosylations or other post-translational use of isotope labeling) quantitation of peptides resolved
modifications (PTMs) of interest [32, 33]. Furthermore, by the nano-LC separation, based on principles such as
proteins can be electrophoretically transferred from the spectral counting or peak signal integration. However,
gels to membranes for subsequent immunoblotting ana- this type of quantitation is especially suited for relative
lysis using antivenoms [29, 30, 34]. comparisons of identical components among different
On the other hand, although 2DE analysis presumably samples, rather than for absolute estimations within a
reflects better the venom protein complexity in a single sample [36]. The fact that different peptides intrinsically
image than any other protein separation approach, limi- present large variations in their ionization efficiency is
tations inherent to the gel-based strategies for proteomic an obvious obstacle for absolute abundance estimations.
profiling have also been pinpointed. First, only proteins Furthermore, factors such as the multidomain construction
and large peptides are retained in the electrophoretic of some snake venom protein families (e.g., metalloprotein-
gels, while peptides smaller than 2-3 kDa are lost. Short ases, multimeric complexes, etc.) introduce uncertainties in
peptides can be abundant components of some snake the assignment of tryptic peptides to intact parent mole-
venoms, and may display relevant bioactivities [35]. An cules if these are digested together.
additional drawback of the gel-based strategies is the On the other hand, some features of the LC-based
limited dynamic range of protein concentrations in the strategies make them an attractive option for the study
original sample that can be resolved electrophoretically of snake venoms, such as the simple preparation of sam-
into non-overlapping spots, which also bears a relation- ples, and the high-speed/high-throughput, automated pro-
ship to the maximal limits in sample loads of the 2DE cessing of the LC-MS/MS runs, together with the deep
technique. Finally, some proteins exhibiting extreme pI’s, detection of trace protein components. Notwithstanding,
close to the limits of the pH gradient used in the first di- these powerful strategies have thus far provided most often
mension isoelectrofocusing step, or unstable proteins qualitative information on venom composition. It should
with a tendency to aggregate or precipitate, may be lost, be stressed that relative protein abundances reported in
or produce inconvenient ‘streakings’ that affect the over- some studies based on this analytical pipeline [37, 38], as
all resolution. It is also possible that single spots might well as on the 2DE workflow [39, 40], correspond to
contain two or more proteins, and this is particularly ‘frequency of identification’, or ‘percentage of the protein
evident when MS/MS identification is performed on sequences’, which may not be necessarily equivalent to
high-end, sensitive instruments. Regarding the estima- abundance [41], and may therefore not reflect the actual
tion of protein abundance, 2DE images can in principle quantitative distribution of components in the venom.
be analyzed by densitometry. However, such quantitation Thus, in all peptide-based quantitation techniques, the
can be complex, and is generally considered less reliable assumption is made that protein digestion is complete,
in comparison to the simpler band patterns generated by and that the resulting proteolytic peptides are equally
one-dimensional electrophoresis [17]. detectable by the mass spectrometric technique used
for the analysis.
LC-based proteomic strategies In addition, the assumption ‘one peptide = one protein’
LC-based proteomic profiling strategies (Fig. 1b) rely is obviously not true for proteins with repeat units, or
completely on the chromatographic separation of peptides for highly similar isoforms that share large parts of their
resulting from the proteolytic digestion of the whole amino acid sequences. Moreover, shotgun strategies do
venom sample. Also known as ‘shotgun’ proteomics, in not allow further combinations with appended tech-
this kind of approach an impressive resolution of peptides niques to expand the informative value of the analyses.
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 4 of 12
Further, owing to the fully automated processing of peptide bonds) in the RP-HPLC step, combined with
matching the fragmentation spectra against databases, densitometry scanning of the SDS-PAGE step when
limitations on available information for snake proteins a fraction is resolved into several electrophoretic
become of concern. New algorithms for proteomic ana- bands; and
lysis are achieving impressive progress and efficiency in 
 by performing SDS-PAGE of venom fractions under
the automated de novo sequencing of peptides from MS/ both reducing and non-reducing conditions,
MS spectra [42–44], and this may counterbalance the covalently-linked subunit composition of multimeric
problem of venom proteins database limitations. proteins can be deduced.
Combined LC/gel-based proteomic strategies Regarding the basic equipment for sample decomplexa-
A workflow combining an LC first dimension separation, tion, the venomics strategy requires commonly available
with a one-dimensional electrophoresis (SDS-PAGE) as electrophoresis setup for SDS-PAGE (one dimensional), as
second dimension, was introduced by Calvete et al. [45, 46] opposed to higher cost isoelectrofocusing equipment
who referred to it as ‘snake venomics’. In this approach needed for 2DE. It also requires regular HPLC instru-
(Fig. 1c), venom decomplexation is first performed by ments of analytical scale, in contrast to shotgun LC-based
RP-HPLC on a C18 column at analytical scale, in the strategies which generally use more costly multidimen-
range of 0.5-2 mg of sample load. Resolved fractions sional nano-flow HPLC chromatographs.
are manually collected, and further separated by one- On the side of drawbacks, the venomics workflow in-
dimensional SDS-PAGE, where resulting protein bands volves a more manually-oriented benchwork, and trace
can be excised and in-gel digested, to be finally submit- components are more prone to escape detection, as
ted to MS/MS analysis. Comparatively, this approach is already mentioned. In addition, it has been noted that
slow and requires significant manual work, especially in some large proteins of low abundance in the venom (for
the collection and subsequent processing of chromato- example hyaluronidases), might be difficult to elute from
graphic fractions. Furthermore, protein components that the C18 HPLC columns, and thus could be overlooked in
are present in trace amounts are generally more likely to some cases. Also, although most small and medium-
be overlooked, in comparison to full LC-based strategies, sized venom components can be recovered in a func-
due to the sampling bias of proteins that are more evident tional state from the RP-HPLC separation, a number of
in the chromatographic pattern and the stained gels. larger proteins/enzymes become denatured by the aceto-
However, several advantages of this workflow may nitrile gradients used for the elution, and therefore lose
compensate these potential shortcomings, and altogether their activities, as discussed below.
support its choice when the biological significance of
the results is prioritized over the mere cataloguing of ‘Snake venomics’ as a useful proteomic profiling
proteins: workflow
Currently, proteomic profiles of the venoms from more

 small peptides (or other compounds such as than 200 snake species have been reported in the litera-
nucleosides) are recovered from the RP-HPLC step, ture, and numbers continue to grow. Venoms have been
in contrast to 2DE strategies; studied by a variety of analytical strategies, among them

 loading of the HPLC-resolved fractions onto gels for the ‘snake venomics’ workflow, utilized in the laborator-
SDS-PAGE can be ‘normalized’ or adjusted, aiming ies of both authors, has contributed with a considerable
to obtain protein bands of adequate staining-intensity proportion of the published data. With the purpose of
(for in-gel digestion) even from chromatographic contributing to emerging research groups interested in
peaks that greatly differ in magnitude due to the this subject, a summary of the general conditions for the
dissimilar proportions of components in the initial RP-HPLC separation of crude venoms used in
venom. This normalization is not possible in the many of the venomics studies is presented in Fig. 2.
2DE or LC-based shotgun workflows; The acetonitrile gradient used for elution (Fig. 2) is a

 analytical scale RP-HPLC allows for considerable scaled-down adaptation of the originally described method
venom sample loads, within the milligram range, of 180 min [46] to 90 min [47], but retaining the same
which allows fractions to be recovered in sufficient shape. A significant saving in time and solvents, without
amounts for complementary analyses, both functional compromising resolution and pattern of elution, has been
and immunological, as will be discussed in the observed (unpublished results). Although each laboratory
following sections; usually develops and optimizes its preferred HPLC pro-

 the relative abundances of identified proteins can be tocols, adopting a common method could aid in the
estimated from the integration of peak areas of standardization and comparability of results among dif-
absorbance at 215 nm (absorption wavelength of ferent research groups.
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 5 of 12
Fig. 2 Scheme for RP-HPLC fractionation of snake venoms. A considerable number of snake venomic studies have used the chromatographic
conditions indicated in the diagram. The venom proteins are separated using an analytical (4.6 × 250 mm, particle diameter of 5 μm) reverse-phase C18
column, eluted at a flow rate of 1 mL/min by a linear gradient of water containing 0.1% of trifluoroacetic acid (TFA) (solution A) and 70% acetonitrile
(CNCH3) containing 0.1% TFAa, and the eluate monitored at 215 nm. The timetable for the mixing of these solutions (A, B), and the shape of
the gradient (dashed line) are indicated. As an example, the approximate elution regions for some of the common protein components of
snake venoms are indicated by colored boxes. This procedure has been applied to venoms of a number of viperid and elapid snakes, helping
in the standardization and comparability of results between different laboratories. 3FTx: three-finger toxin; Kunitz: Kunitz-type serine protease
inhibitor; PLA2: phospholipase A2; CTL: C-type lectin; SP: serine protease; CRiSP: cystein-rich secretory protein; NGF: nerve-growth factor;
VEGF: vascular endothelium growth factor; MP: metalloproteinase; LAAO: L-amino acid oxidase; PDE: phosphodiesterase; 5′-NU: 5′-nucleotidase;
HYA: hyaluronidase; PLB: phospholipase B
Antivenomics: the immunorecognition profiling of Antivenomic analyses can reveal which venom proteins
venom antigens are strongly, poorly, or even not immunorecognized by a
An important area within snake venom research deals given antivenom, providing valuable knowledge on the rela-
with the development, preclinical testing, and clinical tive immunogenicity of these components in the animal
monitoring of antivenoms used for the treatment of hu- species in which the antidote was produced. Moreover,
man or animal envenomation. These essential antidotes these methods also offer a means for assessing cross-
save thousands of lives every year. The preclinical recognition between particular components in the venoms
characterization of antivenoms has mainly involved as- of different snake species, or intraspecific variations related
says to assess their neutralizing potency against the le- to geographical distribution or age [32, 50–62]. In conjunc-
thal effect of whole venoms in animal models, usually tion with venomics data, antivenomics represents a signifi-
mice, although often the neutralization of other relevant cant step forward in the preclinical characterization of
venom activities is reported as well [48]. antivenoms, bringing further information to support
The introduction of proteomic analyses applied to decisions on the selection of venom immunogens for
snake venoms has opened new opportunities to deepen the production of improved antivenoms, for example.
our knowledge on the detailed immunorecognition of It must be stressed, however, that antivenomic analyses
venom components by antivenoms, an area that has been are restricted to the immunorecognition of venom anti-
referred to as ‘antivenomics’ [49]. Taking advantage of the gens and, sensu stricto, this does not automatically
thorough compositional information on venoms provided imply neutralization of their toxic effects. For the pur-
by proteomic tools, methods have been devised to assess pose of the latter, neutralization assays remain the gold
their individual component recognition by antibodies, standard. Nevertheless, when dealing with polyclonal
using a variety of immunoassays (Fig. 3). antibodies, immunorecognition is often a good predictor
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 6 of 12
Fig. 3 Antivenomic analytical strategies. A schematic representation of immunological approaches that have been combined with proteomic
analysis of snake venoms, aiming to assess the immunorecognition of venom components by antibodies present in a given antivenom.
a Immunoblotting, performed on electrotransferred membranes from two-dimensional gel electrophoresis (2DE) venom separations, identifies
spots that are immunorecognized by the antivenom, in an essentially qualitative way. Immunoblotting can also be performed on membranes
from the electrophoresis step (second dimension separation by SDS-PAGE) of the snake venomics strategy (see text and Fig. 1c). b ‘First generation’
antivenomics evaluates the immunodepletion of venom components after addition of antivenom and removal of precipitated immunocomplexes. The
remaining supernatant is analyzed by HPLC and its profile is compared to that of a control venom aliquot. Differences in the chromatographic peaks
between the antivenom-treated venom and the control venom can be quantified by integration of their peak areas, representing the
immunodepletion of recognized components. c ‘Second generation’ antivenomics evaluates the venom components that are captured by
an antivenom that has been covalently linked to beads, following the principles of immunoaffinity chromatography. Whole venom is incubated with
this matrix and the unbound components are collected. After washing out the non-binding venom components, a change in pH elutes the bound
venom fraction. Both samples are finally analyzed by HPLC, and their profiles are compared to that of a control sample of venom. Quantitative
estimations of the degree of immunorecognition of components are performed as described for panel b by integration of chromatographic
peak areas [58]. d HPLC/ELISA-based assessment of immunorecognition of venom components by an antivenom, or HPLC/ELISA-based immunoprofiling,
is performed by coating microwell plates with a normalized amount of venom fractions obtained from the HPLC profile of the venom. Then, antivenom
is added to each well and the bound antibodies (Ab) are detected by conventional ELISA
of neutralization. Therefore, antivenomic analyses provide immobilized onto the beads of an affinity matrix, which
highly valuable information to the overall characterization is then used to separate bound from unbound venom
of antivenoms. components. The antivenom-bound or ‘immunocaptured’
The original antivenomics protocol developed in Calvete’s venom fraction is eluted by a change in pH, and then both
laboratory [63] was based on the immunoprecipitation of fractions, as well as non-venom specific IgG and matrix
antigen-antibody complexes formed by mixing of venom controls, are analyzed by RP-HPLC to compare their pro-
and antivenom in fluid-phase (Fig. 3b). Venom antigens are files and quantify the degree of immunorecognition of
depleted from the supernatant if recognized by antibodies, each venom component.
and the RP-HPLC profile of the supernatant can then be Immunoaffinity-based antivenomic analyses require a
compared to that of a control venom sample in order to careful control of all chromatographic conditions and a
assess the degree of immunodepletion of each peak. A sec- standardization of parameters for each particular anti-
ond generation antivenomics protocol was developed venom/venom system. Inadequate proportions of venom
(Fig. 3c), switching from a fluid-phase immunoprecipita- and antivenom in the system might strongly affect the
tion to a solid-phase interaction provided by immuno- results due to the saturation of binding sites in the
affinity chromatography [64]. Antivenom is covalently solid-phase matrix [65]. In addition, potential losses that
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 7 of 12
may occur during the recovery of bound and unbound quantitative calculations, as done in immunoaffinity-based
venom fractions must be taken into consideration to antivenomics.
avoid introducing errors in the quantitative comparison Independently of the immunological methods adopted
of the subsequent HPLC profiles. On the other hand, in the different analytical formats (Fig. 3), the possibility
the smoother baseline in chromatograms of the affinity of combining the proteomic profile of venoms with the
column allowed better resolution and more accurate immunorecognition of its components by antivenoms,
quantification of the antivenomic outcome than the has provided a considerable increment in the inform-
original immunodepletion protocol. Furthermore, ad- ative value of studies in this field. By such combination
vantages of the second generation antivenomics are the of methods, information on antigenicity and immunore-
possibility of analyzing F(ab’)2 antivenoms and the reus- cognition can be added to the detailed cataloguing and
ability of the affinity columns. These features contribute to abundance estimation of venom components (Fig. 4).
the generalization, economy and reproducibility of the
method. Toxicovenomics: unmasking the villains among
The second-generation antivenomic strategy outlined the crowd
above has been used most often in recent characteriza- Venoms are relatively complex secretions mainly com-
tions of antivenoms [66–68]. Additional types of immuno- posed of proteins and peptides which, by common sense,
assays have also been combined with venomic analyses in would be expected to display the major toxic activities of
order to evaluate the specificity of antibodies present in an the venom. However, not necessarily every component
antivenom toward particular venom proteins. Immuno- present in a venom must be toxic, or not necessarily be
blotting (Fig. 3a) can be performed on membranes electro- toxic for every animal, whether experimental subject or
transferred from 2DE venom separations, incubated with natural prey. In addition, it seems reasonable to assume
antivenom, and developed for detection of bound anti- that some of the components may have a predominant
bodies [29, 34, 69]. In another immunoblotting strategy, role over others in the overall toxic effects of the venom.
the SDS-PAGE patterns of all venom fractions previously Recent studies have taken advantage of the known
separated by RP-HPLC (following the ‘snake venomics’ power of proteomic tools in dissecting and identifying
protocol), can be electrotransferred and similarly devel- the detailed composition of snake venoms, by combining
oped with antivenoms [47, 63, 70–72]. Adequate parallel this information with diverse functional assays (Fig. 4).
controls of non-immune sera matching the species from Such combined strategy was first referred to as ‘toxi-
which antivenoms are produced are indispensable in all of covenomics’ at the 18th World Congress of the Inter-
these immunological techniques. Immunoblotting-based national Society on Toxinology (IST) held in Oxford in
methods in the assessment of antivenom specificity have 2015 [80].
two important limitations: (a) results are essentially quali- The essence of the toxicovenomics approach lies in
tative; and (b) some epitopes of venom components can screening the RP-HPLC resolved profile of venom frac-
be disrupted due to the denaturing effect of SDS detergent tions provided by the venomics workflow, for specific
during either the 2DE or one-dimensional SDS-PAGE toxic activities. For example, screening for lethality to
procedures. rodents would identify which venom components may
A fourth approach for the antivenomic assessment play a role in the potentially lethal effects in humans, or
of immunorecognition of venom components is based screening for myotoxicity would identify components
on enzyme-immunoassays such as the ELISA format relevant to the skeletal muscle tissue damage induced by
(Fig. 3d). Protein peaks resolved by the RP-HPLC step some venoms in clinical envenomation, and so forth.
of the venomics protocol are collected, normalized for Thus, as a third pillar for a broader, more integrative
concentration, and coated onto microwell plates. Then, view of snake venoms, toxicovenomic characterizations
the presence of antibodies toward each chromatographic add valuable information of biological and medical
fraction, in a given antivenom, can be determined by significance.
ELISA [73–79]. Although this combined HPLC/ELISA A key concept related to toxicovenomic analysis was
immunoprofiling approach provides a general view of introduced by Laustsen et al. [81], which seeks to iden-
the immunorecognition/immunogenicity of the different tify those components of a given venom that are mainly
venom components along its full chromatographic elution responsible for its toxicity, for example its lethal effects
profile, it is also not exempt from limitations. Among these, on mice: the ‘Toxicity Score’ (TS). By combining data on
epitopes of venom antigens may become potentially altered the identity, abundance, and lethal potency (median lethal
by the solid-phase coating. Also, the intensity of absorbance dose; LD50) of each venom fraction, a TS is obtained by
signals provided by different venom fractions are influenced dividing its estimated relative abundance (% of total pro-
by a number of factors, such as epitope density and anti- teins) by its LD50 value. Then, it is possible to rank venom
body saturation, thus precluding the possibility to perform components in terms of their functional predominance to
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 8 of 12
Fig. 4 Evolution of analytical strategies in the characterization of snake venoms by proteomic tools, used in combination with appended
methodologies. Initial proteomic studies on venoms essentially focused on the qualitative cataloguing of components. The introduction of the
snake venomics strategy led to a valuable increase in the informative value of these analyses, by providing an estimation of the abundances of
venom components. In combination with antivenomics, the immunogenicity of venom components can be inferred by evaluating their
recognition by antibodies present in a given antivenom. A third dimension in the characterization of venoms is provided by a combination with
toxicovenomics, which evaluates the toxic activities of components. Altogether, these combined strategies increase the informative value of
studies characterizing venoms by disclosing their composition (venomics), immunorecognition (antivenomics) and toxicity (toxicovenomics)
the overall effect of the venom, and therefore identify Hence, the toxicovenomic characterization of a venom is
those that play most relevant roles. also of great relevance in the field of the evolutionary ecol-
The combination of toxic potency and abundance into ogy of the organisms that produce the venom; and vice
a score allows a better view of the relevance of particular versa, the identification of toxins bearing the highest evolu-
toxins in envenomation, as compared to toxic potency tionary pressure is also of great relevance for the design of
alone [81]. This concept was developed with the purpose more effective antidotes.
of identifying which venom components should be tar- Although the addition of toxicovenomic evaluations to
geted by novel neutralizing agents under development, proteomic data appears in principle a simple concept, in
such as recombinant human antibodies or synthetic pep- practice there are still several important limitations to
tide inhibitors [82]. Several investigations on elapid snake overcome. Among these is the fact that medium- to
venoms have succeeded in pinpointing the main targets to large-size enzymes/proteins may easily become denatured
be inhibited by using this experimental ‘toxicovenomics’ by the RP-HPLC conditions used to separate venoms. Me-
approach [73, 74, 78, 79]. talloproteinases, for example, are inactivated by organic sol-
Recent studies on the proteomic characterization of vents commonly used in reversed-phase chromatography,
venoms are increasingly combining identification data and this has largely precluded the application of toxicove-
with functional assays of particular components, to gain nomic strategies based on RP-HPLC to the venoms from
deeper insights from the medical and biological perspec- viperids, which are generally rich in such enzymes. In the
tives [57, 83–85]. The TS is conceptually identical to the case of elapids, since many of them have very low propor-
‘lethal neurotoxicity coefficient’ (LNC) defined as the ratio tions of metalloproteinases (i.e., < 5% of the total prote-
between the average LD50 and the crotoxin + crotamine ome), toxicovenomic screenings have succeeded owing to
relative abundance (% of the total venom proteins) [50]. the fact that their major components, such as three-finger
The LNC was introduced to provide a quantitative measure toxins, phospholipases A2, Kunitz-type serine protease in-
of the evolutionary pressure towards gain of neurotoxicity hibitors, etc., withstand the organic solvents and retain full
and lethal activities of the venom of C. durissus snakes to- functionality. However, there is a need to develop better-
wards rodents, which evolved along the North-South axis suited chromatographic methods under native conditions,
of the invasion of South America, coincident with the evo- using aqueous buffers, with a resolution capable of parallel-
lutionary dispersal pattern of the Neotropical rattlesnakes ing that of RP-HPLC, in order to expand the applicability
[50]. This underscores the view that toxins bearing the of functional screenings to the venoms of viperids.
highest toxicity score may represent the same proteins re- The resolution of size-exclusion chromatography
sponsible for the evolutionary adaptive potential of venom. (SEC)-HPLC columns is still comparatively low, and the
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 9 of 12
use of ion-exchange HPLC-based columns limits the that venoms enclose, by developing beneficial applications,
possibility to separate all venom components (acidic and thus literally turning poisons into potions [92, 93].
basic) in a single run. Possibilities to combine different Although it is hard to predict the future directions of a
non-denaturing HPLC-based separations need to be ex- rapidly changing field dominated by technological ad-
plored in order to expand the applicability of toxicove- vances – such as proteomics – it is likely that venomics
nomic assessments to a broader range of snake species. will seek improved quantitative methods to calculate
A second consideration about toxicovenomic evalua- more accurately the abundance of venoms components
tions concerns the possibility of having different venom [94]. Further, venomics will benefit from the rapidly in-
components that act synergistically, i.e. where each of creasing availability of genomic and transcriptomic data,
them separately may be weakly toxic, but together may to evolve its resolution power from a protein-family
result in a strong enhancement of a toxic effect, as iden- level, to a locus-resolution level, even encompassing pro-
tified, per instance, in Micropechis ikaheka venom [86]. teoform variability [94]. Regarding antivenomics, the fu-
Venoms whose sum of TS values of all separated frac- ture should bring further refinements and application of
tions results in a significantly lower value in comparison techniques for determining the fine specificity of anti-
to the TS of the unseparated material, should be sus- bodies that recognize and neutralize toxins, identifying
pected to enclose synergistic components [81]. their most relevant antigenic determinants through
A final consideration on toxicovenomic assessments strategies such as epitope mapping using sets of overlap-
relates to the choice of model for the evaluation of tox- ping synthetic peptides [95–97], including the recently
icity. It is known that some venoms may be highly toxic reported use of high-density peptide microarray technol-
to certain types of animals, but not to others, and the ogy for such purpose [98]. Toxicovenomics, still in its
concept of ‘taxon-specific toxins’ has been demonstrated infancy, will need to cope with limitations and chal-
in various studies [87–89]. As a general rule, experiments lenges already discussed, on the resolution of native
evaluating toxic activities with the purpose of investigating chromatography strategies, and the development of per-
biological aspects, such as evolutionary or ecological in- tinent bioassays, preferably in vitro.
quiries, should consider the use of species reported to be Currently available methods in all these three areas
natural prey for the particular venomous snake. Instead, that aim at an integrative view of the venoms are cer-
for the study of applied aspects of venoms that are medic- tainly not free of limitations and challenges. There is
ally oriented, such as the development of antidotes or the plenty of space for ingenious improvements, welcoming
study of pathological features experimentally induced by opportunities and ideas to develop and validate better
the toxins, mice or other mammalian models would be procedures than the currently available. As earlier stated
more pertinent, owing to their closer relatedness to by the authors [99], a bright future for integrative
humans and the ease of controlling all relevant variables venomics is on the toxinology horizon.
to normalize the results.
Abbreviations
2DE: Two-dimensional gel electrophoresis; LC: Liquid chromatography;
LD50: Median lethal dose; LNC: Lethal neurotoxicity coefficient; MS: Mass
Conclusions spectrometry; MS/MS: Tandem mass spectrometry; Mw: Molecular weight;
Undoubtedly, the application of proteomic tools to snake pI: Isoelectric point; PTM: Post-translational modifications; SDS-PAGE: Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis; TFA: Trifluoroacetic acid;
venom research has resulted in an unprecedented expan- TS: Toxicity Score
sion of knowledge on their overall composition, in a grow-
ing number of species. Here, we have briefly discussed Acknowledgments
some recent developments in this area, highlighting how We thank our many colleagues at the laboratories in Valencia and San Joséwho have contributed to venomics studies during the past 8 years, as well
strategies have evolved from the mere cataloguing of as many collaborators who have provided valuable samples for such studies.
venom components (proteomics/venomics), to a broader
exploration of their immunological (antivenomics) and Funding
Research at IBV-CSIC was supported by grant BFU2013-42833-P from the
functional (toxicovenomics) characteristics (Fig. 4). Ministerio de Economía y Competitividad, Madrid, Spain. Research at ICP-UCR
Altogether, the combination of these complementary was supported by Vicerrectoría de Investigación, Universidad de Costa Rica.
strategies is helping to build a broader view of the dan-
gerous protein cocktails produced by venomous snakes, Authors’ contributions
All authors conceived and wrote this review, and approved the final
responsible for thousands of deaths every year around manuscript.
the globe. Such knowledge on snake venoms should
provide better opportunities to cope with the great suffer- Competing interests
ing inflicted on the individual and social levels [90, 91]. The authors declare that they have no competing interests.
And, on the other hand, this knowledge should allow us to Consent for publication
discover and explore the formidable bioactive molecules Not applicable.
Lomonte and Calvete Journal of Venomous Animals and Toxins including Tropical Diseases  (2017) 23:26 Page 10 of 12
Ethics approval and consent to participate 21. Aird SD, Watanabe Y, Villar-Briones A, Roy MC, Terada K, Mikheyev AS.
Not applicable. Quantitative high-throughput profiling of snake venom gland
transcriptomes and proteomes (Ovophis okinavensis and Protobothrops
flavoviridis). BMC Genomics. 2013;14:790.
Publisher’s Note 22. Margres MJ, McGivern JJ, Wray KP, Seavy M, Calvin K, Rokyta DR. Linking
Springer Nature remains neutral with regard to jurisdictional claims in the transcriptome and proteome to characterize the venom of the
published maps and institutional affiliations. eastern diamondback rattlesnake (Crotalus adamanteus). J Proteomics.
2014;96:145–58.
Author details
1 23. Castoe TA, Braun EL, Bronikowski AM, Cox CL, Rabosky ARD, de Koning AP J,Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa
2 et al. Report from the first snake genomics and integrative biology meeting.Rica, San José 11501, Costa Rica. Structural and Functional Venomics Stand Genomic Sci. 2012;7(1):150–2.
Laboratory, Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain. 24. Koepfli KP, Paten B, Genome 10K Community of Scientists, O’Brien SJ. The
Genome 10K Project: a way forward. Annu Rev Anim Biosci. 2015;3:57–111.
Received: 24 January 2017 Accepted: 19 April 2017 25. Li S, Wang J, Zhang X, Ren Y, Wang N, Zhao K, et al. Proteomic
characterization of two snake venoms: Naja naja atra and Agkistrodon halys.
Biochem J. 2004;384(Pt 1):119–27.
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