Next-generation snake

venomics: protein-locus
resolution through venom
proteome decomplexation
Expert Rev. Proteomics Early online, 115 (2014)
Juan J Calvete
Instituto de Biomedicina de Valencia,
Consejo Superior de Investigaciones
Cient ficas, Jaime Roig 11, 46010
Valencia, Spain
Tel.: +34 963 391 778
Fax: +34 963 690 800
jcalvete@ibv.csic.es
Venom research has been continuously enhanced by technological advances. High-throughput
technologies are changing the classical paradigm of hypothesis-driven research to
technology-driven approaches. However, the thesis advocated in this paper is that full proteome
coverage at locus-specific resolution requires integrating the best of both worlds into a protocol
that includes decomplexation of the venom proteome prior to liquid chromatographytandem
mass spectrometry matching against a species-specific transcriptome. This approach offers the
possibility of proof-checking the species-specific contig database using proteomics data.
Immunoaffinity chromatography constitutes the basis of an antivenomics workflow designed to
quantify the extent of cross-reactivity of antivenoms against homologous and heterologous
venom toxins. In the authors view, snake venomics and antivenomics form part of a
biology-driven conceptual framework to unveil the genesis and natural history of venoms, and
their within- and between-species toxicological and immunological divergences and similarities.
Understanding evolutionary trends across venoms represents the Rosetta Stone for generating
broad-ranging polyspecific antivenoms.
KEYWORDS: affinity chromatography antivenom antivenomics mass spectrometry snakebite envenoming
snake venom venomics
The evolutionary link in venom research
Venoms represent an adaptive trait and an
example of both divergent and convergent evo-
lution [1]. The ecological advantages conferred
by the possession of a venom system are evi-
dent from the extraordinarily diverse range of
animals that have evolved venoms for hunting,
defense efficiency or competitor dissuasion.
Every ecosystem on Earth supporting life con-
tains venomous organisms, and the extant suite
of venomous animals includes over 170,000
species throughout all major phyla of the evo-
lutionary tree of the animal kingdom from
ancient cnidarians through annelids, nemer-
tines, echinoderms, mollusks, arthropods and
chordates.
Snakes are represented on earth today by some
3150 species, which represent a single massive
diversification event that occurred after the K-T
boundary at the time of the extinction of the
dinosaurs [2]. Extant snakes are found throughout
most of the world (including the oceans), except
for a few islands, frozen environments and high
altitudes. Venomous snakes, represented by
600 or so species within Viperidae, Elapidae and
Atractaspidinae, have developed muscularized
venom glands and tubular front fangs [3]. In
addition, venom in an increasing number of
rear-fanged snakes (Serpentes, superfamily Colu-
broidea) has also been documented and studied
at proteomic and transcriptomic levels ( [49] and
references therein). Furthermore, the demonstra-
tion that snakes and all lizard lineages possessing
toxin-secreting oral glands (Helodermatidae,
Anguidae, Varanidae and Iguana) form a
clade [10] for which the previously suggested
name Toxicofera [11] (Greek for those who bear
toxins) was adopted, provided overwhelming
support for a single, early origin of the venom
system in lizards and snakes [10]. The proposed
common ancestor of venomous squamate reptile
species may have originated at the base of the
colubroid radiation, approximately 6080 million
informahealthcare.com 10.1586/14789450.2014.900447 2014 Informa UK Ltd ISSN 1478-9450 1
Review
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years ago [2,912]. Though the role of venom of lizards and colu-
brids in their feeding behavior, adaptive ecology or defence from
predators remains a matter of study and debate, the venom systems
of these taxa represent an untapped source of novel-led com-
pounds potentially useful in drug design and discovery. In addi-
tion, despite the venoms of front-fanged snakes provide model
systems for investigating predatorprey interactions, the molecular
bases for evolutionary and ecological adaptations, the generation of
chemical and pharmacological novelty [13] and strategies for the
knowledge-based design of antivenoms to reduce the burden of
the neglected pathology of snakebite envenoming that kills or
maims thousands of healthy individuals every year [14,15], only
recently has the research on venoms began to flourish [13,16]. Dur-
ing the last decade or so, advances in instrumentation and high-
throughput methodologies have fueled an expansion of the scope
of biological studies and strategies for assessing the toxin composi-
tion of snake venoms (snake venomics), directly (through
proteomics-centered approaches) or indirectly (via venom gland
transcriptomics and bioinformatic analysis) in a relatively rapid
and cost-effective manner. For an overview of snake lineages for
which proteomics or transcriptomics venom composition data
have become available, please consult [6] and Table 1 of [16]. Snake
venomics on a few more taxa have been reported since the publica-
tion of this review [16] and can be consulted addressing the NCBIs
PubMed database [17]. More recently, the genomes of the worlds
longest venomous King cobra (Ophiophagus hannah) and the non-
venomous Burmese python (Python molurus bivittatus) have been
sequenced [18,19], and other snakes have been targeted for genome
sequencing [20]. These studies provide an insight into the biology
of the venom in snakes and allow the understanding of the evolu-
tion of venom genes at the genome structural level. The Burmese
python and King cobra studies represent a significant addition to
systems genomics and the foundation of the field of comparative
snake genomics. Thus, comparison between these genomes has
revealed dynamic evolution and adaptation in the snake venom
system, which seemingly occurs in response to an evolutionary
arms race between venomous snakes and their prey. These adapta-
tions include the massive and rapid expansion of gene families that
produce venom toxins that correlate directly with their functional
importance in prey capture.
Refined during eons of biological evolution, snakes produce a
staggering number of compounds in their venoms with therapeutic
and pharmacological potential, a fact that is attracting increasing
interest in academic, industrial and medical arenas [21]. Their high
molecular specificity and potency have long made venom a promis-
ing source of novel compounds for use in drug design and develop-
ment. More than 30 years ago, the US FDA approved the first
venom-derived drug, a therapy for hypertension, called Captopryl

,
developed from bradykinin-potentiating peptides isolated from
venom of the South American pit viper Bothrops jararaca [21]. Ven-
oms exhibiting anticoagulant properties are extensively studied for
possible medical applications, and a handful of venom-derived
drugs, such as Tirofiban

, Eptifibatide

, Batroxobin

, has already
been approved for cardiovascular diseases [21]. The pipeline from
fang to pharmacy is expanding with the discovery of peptides
isolated from black mamba venom that block neuronal acid-sensing
ion channels that play a key role in the pain pathway [22].
The basic and applied aspects of venom research represent two
sides of the same coin: understanding the principles governing the
evolution of venomous systems is not only of medical relevance, for
understanding the molecular basis for adaptive variations in snake
venom phenotypes is of outmost importance for improving current
antivenoms, and also for learning how to use deadly toxins as thera-
peutic agents. Developing the full potential of venom research
requires the integration of data across the biological system within
the frame of an evolutionary hypothesis. Thus, although initially
conceived as a technological platform for the proteomics characteri-
zation of the chemical space [23], natural history and immunoreactiv-
ity of snake venoms with homologous and heterologous antivenoms
[24,25], the inclusion of the evolutionary hypothesis for clustering ven-
oms based on within- and between-species shared traits and trends,
has catalyzed the revival of snake venomics as a conceptual frame-
work for the comprehensive analysis of venoms [16]. This review
focuses on the concepts and technological alliances that guided this
transition from the mere inventory of toxins to biology and discusses
foreseeable future developments in the field.
Decomplexing the venom proteomes: an opportunity
to quantify the relative abundances of the venom
components
Snake venom toxins originated by duplication of ordinary genes
and the accidental expression of the duplicated copies in the
venom gland, followed by amplification of the co-opted genes to
multigene families via a birth and death mode of evolution [26],
and the rapid diversification of these families by extensive neo-
functionalization of some copies and the transformation into
pseudogenes of nonfunctional forms [1,2,2729]. As a result of this,
after approximately 100 million years of evolution, venoms of
extant snakes comprise moderate complex mix of <10 [30,31] to
more than 50 [32,33] toxins, which largely belong to a few major
protein families, ranging from only 2 to about 20 [32,34]. However,
the number of proteoforms (sensu [35]) in the venom can be signif-
icantly greater due to post-translational modifications, proteolytic
processing and oligomerization [3639]. In a neutral evolutionary
scenario, interspecific differences in venom composition should
be closely related to the degree of phylogenetic divergence
between species. A strong association between venom composition
and phylogenetic relationships of species has been postulated
from observations that broad-scale patterns of venom composition
mirror deep phylogenetic relationships among Elapidae snakes
(reviewed in [40]). However, the proteomic and transcriptomic
diversity of venom in elapid snakes have not been intensively
investigated, and significant intragenus and intergenus variations
in venom profiles have been reported in Australian elapids [41,42].
Also, in Viperidae, venom variation appears to be evolutionarily a
highly labile trait even among very closely related taxa [43]. In
terms of practical applications, the low correlation between phy-
logeny and venom variation argues against using phylogeny as the
sole criterion for selecting a venom mixture in antivenom produc-
tion strategies; proteomics-guided identification of the composition
Review Calvete
doi: 10.1586/14789450.2014.900447 Expert Rev. Proteomics
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of and immunoreactivity trends across venoms represents the
approach of choice.
The development of sample preparation protocols (nano-uPLC,
multidimensional-high performance liquid chromatography
(HPLC), 2D electrophoresis [2DE]) coupled to soft ionization
(matrix-assisted laser-desorption ionization and electrospray ioniza-
tion) mass spectrometry has been pivotal to get an accurate picture
of the startling complexity of venoms [44]. Different approaches
have been applied to unlock the peptide and protein composition
of a number of snake venoms including shotgun proteomics (also
known as discovery proteomics) and hyphenated separation tech-
nologies [36,37]. In a shotgun data-dependent acquisition (DDA)
method, the first scan is a survey scan (full MS scan) where the
precursor ions are isolated and subsequently activated. The
obtained fragments are analyzed in a second stage (data-dependent
scan) of mass spectrometry. In many sample types, including ven-
oms, the complexity and dynamic range of compounds can be
very large. This poses challenges for the traditional data-dependent
workflows, requiring very high-speed tandem mass spectrometry
(MS/MS) acquisition to deeply interrogate the sample. Data-
independent acquisition (DIA) strategies have been used to
increase the reproducibility and comprehensiveness of data collec-
tion. In DIA mode, an expanded mass isolation window is stepped
across a mass range covering the mass-to-charge distribution of
peptides, and all ions present at a given time are activated and dis-
sociated without selection. In this case, instead of the serial
collision-induced dissociation (CID) of peptide ions, parallel CID
of ion mixtures take place in. A large mass range can be interro-
gated in an liquid chromatography (LC)MS time frame because
of the larger mass steps. To assign subsequent fragments to correct
precursors, data are continuously acquired by alternating between
high (precursor filtering) and low (dissociation and fragment filter-
ing) voltage potentials [45]. This strategy is also called LCMS
E
[46]. Another variant of the emerging class of DIA workflows is
MS/MS
ALL
with Sequential Windowed data-independent Acquisi-
tion of the Total High-resolution Mass Spectra (SWATH
TM
-
MS) [47]. In SWATH-MS mode, a 25 amu window is transmitted
through the MS1 analyzer into the collision cell. MS2 data are
acquired by repeatedly cycling through 32 consecutive 25 Da
precursor isolation windows (swaths), which cover the full
4001200 m/z range and monitoring all fragment ions. The high
resolution of MS2 spectra (10 p.p.m.) ensures the specificity of
peptide identifications. For a comparative overview of the MS
instrumental principles of conventional shotgun proteomics and
SWATH MS analysis, refer [48].
Tryptic digestion of the whole venom followed by LCCID
MS/MS, no matter whether they are acquired by DDA or DIA
mode, allows the identification of the toxin classes present in the
venom but does not inform about the quaternary structure of indi-
vidual toxins. In addition, a limitation inherent to this methodol-
ogy is its inability to distinguish among protein family members
exhibiting high levels of sequence redundancy. Further, quantifica-
tion by label-free spectral counting of the relative abundances of
venom components based on the information provided by the
shotgun MS data set on the types, number and ion intensities of
the different peptide ion spectra associated with each protein by
MS/MS, requires correction for differential MS detectability of the
contributing peptides (i.e., due to sequence properties that affect
peptide ionization) and protein size (larger proteins contribute
more peptides) or the addition of proteotypic peptides.
2D electrophoretic analysis provides a more realistic view of
venom complexity. However, small peptides are essentially lost,
and colorimetric quantification of individual toxins or toxin fami-
lies is not straightforward. Thus, despite the existence of sophisti-
cated software tools, 2DE gel image analysis still remains a serious
bottleneck, which traditionally involves a preprocessing step to sup-
press noise, correct background and remove artifacts; spot bound-
aries delineation; and expression quantification. The latter step is
done by estimating the relative spot volume: V
spot_i
/
P
V
spots
in the
2DE gel = mass [in SI units, g]% of spot
i
. On the other hand, frac-
tionation of venom components by chromatographic methods
(usually reversed-phase HPLC) prior to MS analysis (FIGURE 1; step
1) is a powerful approach for proteome decomplexation, and in
conjunction with apparent molecular mass determination by SDS
PAGE under reducing and nonreducing conditions (FIGURE 1; step 2)
provides a true appreciation of both the proteolytic processing and
quaternary structure of the venom proteins [23]. Reversed-phase
HPLC represents the method of choice for the fine separation and
quantitative recovery in a single run of peptides (0.47 kDa) and
proteins (7150 kDa) commonly present in snake venoms. In
addition, the fact that the chromatographic fractions are obtained
in a solvent compatible with downstream mass spectrometric
analysis, allows the (on-line or off-line) determination of the
accurate molecular mass of the native venom components (FIG-
URE 1; step 3) and their number of reduced (sulphydryl groups)
and oxidized (disulfide linkages) cysteine residues [23]. The
binomial molecular mass and number of disulphide bonds is
a toxin-specific parameter that discriminates between most
toxin classes found in snake venoms [23].
An important challenge in the characterization of venoms is
the inability of any single method to analyze all unique pro-
teins in the venom proteome. However, the need to decomplex
the venom proteome represents also an opportunity to quanti-
tate the relative abundances of the different venom compo-
nents. Thus, by monitoring the reversed-phase column eluate
at the absorbance wavelength of the peptide bond, 215
220 nm, peak area integration allows an estimation of the rela-
tive abundances (expressed as percentage of the total venom
proteins) of the different chromatographic fractions (FIGURE 1;
step 4). According to the LambertBeer Law, A = ecl; (where
e = molar extinction coefficient [M
-1
cm
-1
], c = concentration
[M], and l = path length [cm]). Expressed in this form, the
extinction coefficient allows for estimation of the molar concen-
tration of a solution from its measured absorbance: A/e = molar
concentration. The calculated figures (%A of component i) cor-
respond thus to the mol% of peptide bonds in component i. The
relative contributions of different proteins eluting in the same chro-
matographic fraction can be estimated by densitometric scanning
after SDSPAGE gel analysis (FIGURE 1; step 2). These figures should
closely match transcriptomic-based measurements of the contents
Next-generation venomics Review
informahealthcare.com doi: 10.1586/14789450.2014.900447
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Review Calvete
doi: 10.1586/14789450.2014.900447 Expert Rev. Proteomics
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of venom toxin transcripts, if the number of reads mapping to a
particular transcript are used as a measure of its abundance [49].
However, to estimate the relative contribution of each toxin or
toxin family as protein molecules/100 molecules of total venom
proteins, the molar percentages of peptide bonds of each family
should be normalized for the number of peptide bonds (amino
acids) in the full-length sequence of a representative member of the
protein family. This way of expressing the relative concentrations of
toxins in the venom allows the direct comparison of proteomic and
transcriptomic data if the relative expression of a given toxin pro-
tein (family) (mol%) is calculated as the number of reads assigned
to this protein (family) (Ri) normalized by the length (in nucleoti-
des [nt]) of the reference transcript sequence (ntREF) and expressed
as the percentage of total reads in the transcriptome (Sreads): mol
% toxin (family) i = %[(Ri/ntREF)/Sreads) [32].
It must be stressed that quantitative venomic approaches only
yield relative, not absolute, toxin quantification. The best
approach currently available for absolute quantitation of a particu-
lar venom component in venom involves the generation of stan-
dard curves for synthetic stable isotope-labeled proteopeptides
added as internal standards to the sample [5052]. In a typical
Absolute Quantification strategy, the sample is spiked with
defined amounts of isotope-labeled analog(s) of specific proteo-
lytic peptide(s), digested and analyzed by shotgun LCMS.
Quantification can be performed in the MS mode by comparing
the extracted ion signal (peak height or peak area) of the isotope
labeled and the native forms of a given proteotypic peptide. In the
tandem mode (MS/MS), a comparison of ion fragment signals
from standard and native peptides can also be performed for
quantification [41,42]. Absolute quantification of full-length pro-
teins would require a Protein Standard Absolute Quantification
strategy. Although an isotope-labeled equivalent of the full-length
target protein appears to constitute the standard of choice, a cur-
rent limitation for applying the Protein Standard Absolute Quan-
tification method in a quantitative venomics protocol is the
difficulty to produce natural (chemically labeled) or recombinant
(isotope-labeled) venom protein standards.
Species-specific transcriptomics: the Rosetta Stone for
achieving locus resolution in venomics analysis
The presence in the same venom of a diversity of proteins of the
same family but differing from each other in their amino acid
sequences and pharmacological activities reflects the accelerated
adaptive molecular evolution of snake venom toxins via positive
selection [13,53,54]. The occurrence of multiple isoforms within each
major toxin family evidences the emergence of paralogous groups
of multigene families across taxonomic lineages where gene dupli-
cation events occurred prior to their divergence and suggests an
important role for balancing selection in maintaining high levels of
functional variation in venom proteins within populations [55].
Omics analysis of venoms across and between different genera
offers the possibility of understanding the molecular evolution of
toxins, the ecological forces and mechanisms driving venom vari-
ability, and the relationship of the components in the venom and
the pathological outcomes of snake envenomings, thereby paving
the way for developing a new generation of broad-range polyspe-
cific antivenoms that are clinically more effective [14,15].
Biologically motivated questions require genome/transcriptome
annotation. However, in these 10 years of first-generation snake
venomics [16,24,56], high-throughput proteomic studies on snake
venoms have been somewhat hampered by the lack of species-
specific venom sequence databases to match the MS/MS data. Not-
withstanding this drawback, de novo interpretation of high-quality
peptide ion fragmentation spectra (FIGURE 1; step 5), in conjunction
with Basic Local Alignment Search Tool analysis of the MS/MS-
derived amino acid sequences (FIGURE 1; step 6), usually allows the
unambiguous identification of an homolog protein in the current
databases [23,24]. The relative abundance (%) of each protein family
represented in the venom can be computed from the relation of the
sum of the areas of the reversed-phase chromatographic peaks con-
taining proteins from the same family to the total area of venom
protein peaks in the reversed-phase chromatogram (FIGURE 1; step 7).
Incomplete sequence coverage resulting from the low-
throughput venomics approach described above and schematized
in FIGURE 1, steps 17 yields toxin family resolution, being usually
unable to distinguish between different isoforms or proteoforms
of toxin family members due to extensive sequence similarity. To
a certain degree, this is being addressed by the production of
transcriptome databases from snake venom glands. However, the
availability of snake venom gland transcriptomes has increased
slowly since the pioneer work of Ho and co-workers in 1995 [57].
For a list of snake species for which transcriptomic, proteomic
and joint transcriptomic and proteomic analyses have been
reported, please refer Table 1 in reference [16].
The majority of the snake venom gland transcriptomic studies
reported to date involve the low-throughput sequencing of clones
Figure 1. Scheme of the second-generation snake venomics platform. Venom proteins are separated by reversed-phase HPLC (1)
and analyzed by SDSPAGE under nonreduced and reduced conditions (2A), and ESIMS (3). The relative abundance of the proteins
eluting in the reversed-phase separation are quantitated by combination of densitometry (2B) and LambertBeer law (4). Electrophoretic
bands are in-gel digested with trypsin and the peptide ions in the digests de novo sequenced by data-dependent CID-MS/MS (5) followed
by protein family identification via BLAST search (6) or data-independent MS
E
(8) and product ion spectra matching to a species-specific
venom gland transcriptomic database (9). Combining qualitative (protein family or locus identifications) (7) and quantitative (relative
abundances derived from absorption data by the LambertBeer Law, c = A/eL) (4) data allow an accurate overview of the toxin
composition of the venom. Comparison of experimental ESIMS toxin masses (3) and those calculated for the species-specific
transcriptome-predicted full-length proteins (10) is a simple and accurate method for discriminating between protein isoforms whose
amino acid sequences share a high degree of identity.
BLAST: Basic Local Alignment Search Tool; CID: Collision-induced dissociation; LCMS: Liquid chromatographymass spectrometry;
MS/MS: Tandem mass spectrometry; RP-HPLC: Reverse-phase high performance liquid chromatography.
Next-generation venomics Review
informahealthcare.com doi: 10.1586/14789450.2014.900447
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randomly picked from a cDNA library constructed by reverse tran-
scription of the RNA molecules expressed in the venom gland [58].
These partial expressed sequence tags cluster into groups of contig-
uous sequences (contigs), which only occasionally cover the entire
extension of the original RNA molecule [58]. Recently, a significant
effort has been directed at generating comprehensive high-
throughput venom gland transcriptomic databases comprising full-
length toxin-coding transcript sequences through next-generation
sequencing platforms [47,56,59]. Next-generation sequencing tech-
nologies are revolutionizing the field of transcriptomics by rapidly
reducing the time and cost per base sequenced.
The most complete characterizations to date of the genes
expressed in active venom glands have been achieved with Illumina
technology [33,49,59]: using 95,643,958 pairs of quality filtered,
100 bp Illumina reads, Rokyta and co-workers [49] identified
123 unique, full-length toxin-coding sequences in the venom gland
transcriptome of an eastern diamondback rattlesnake (Crotalus ada-
manteus). Subsequent nanospray LC/MS
E
analysis of a whole
trypsin-digested venom sample yielded peptide evidence for 52 of
the 78 unique toxin transcript clusters although only 36 toxins
were unambiguously identified based on unique peptide evi-
dence [33]. Clearly, the availability of species-specific full-length
venom gland transcript sequences as a reference database has greatly
enhanced the efforts of MS-based venom proteomics, circumvent-
ing the need for de novo MS sequencing. However, maximizing
proteome coverage at locus-specific resolution requires the integra-
tion into a single second-generation venomics protocol of the ben-
efits of coupling venom proteome decomplexation (FIGURE 1; steps
1 & 2) and matching the output of DDA or DIA shotgun MS/MS
mass spectrometry against the species-specific transcriptome (FIGURE 1;
steps 8 & 9). For example, through the combination of RP-HPLC
separation, SDSPAGE analysis and MS/MS characterization of
the venom components, Wagstaff and colleagues reported the pres-
ence of a multidomain PIV metalloproteinase in Echis ocellatus [60].
In a shotgun approach, the snake venom metalloprotease (SVMP),
disintegrin-like, cysteine-rich and C-type lectin-like (CTL)
domains, would have been detected separately, and the information
on the quaternary structure of this metalloproteinase, which consti-
tutes about 20% of the venom toxins, is lost. Furthermore, since
the covalent linkage of CTL domains to a PIII-SVMP is a post-
translational modification [6163], the quaternary structure of a PIV-
SVMP is not reflected in the transcriptome. The key to identifying
this class of SVMPs is as simple as an SDSPAGE analysis, under
nonreducing and reducing conditions, of the RP-HPLC-isolated
venom fractions, followed by MS/MS identification of domain-
specific peptides in the nonreduced protein band [60].
The identification of highly homologous proteins eluting in dif-
ferent chromatographic fractions or migrating differentially on
SDSPAGE may indicate the coexistence of protein isoforms in
the venom sample. Accurate determination of the molecular
mass (FIGURE 1; step 10) of protein isoforms whose amino acid
sequences share a high degree of identity may be key to identify
the mRNAs that encode them in a species-specific transcriptome
[51,6466]. Linking transcriptome and proteome by mass profiling
are particularly useful in the case of toxin families such as
disintegrin, phospholipase A
2
(PLA
2
), three-finger toxin, snake
venom metalloproteinase of class PI (PI-SVMP), CTL, cysteine-
rich secretory protein, Kunitz-type inhibitor, cystatin, ohanin, b-
defensin-like myotoxin (refer Table 1 in [16]), in which the only
documented post-translational modification is the formation of
disulfide bonds. In a recent paper, Conlon and colleagues [66] iden-
tified transcripts encoding cytotoxic PLA
2
molecules in species-
specific transcriptomic databases using a combination of electro-
spray ionizationMS measurement of the masses of the native
HPLC-isolated proteins and LCMS
E
analysis of tryptic digests.
Venoms from a number of terrestrial and marine elapids are rela-
tively simple, comprising distinct repertoires of toxins typically
belonging to two major protein families, PLA
2
and short and long
three-finger toxins [4,34,6773]. LC separation of venom components
coupled to mass spectrometry fingerprinting is a very simple and
accurate method for quantifying and bridging the gap between
proteome and transcriptome. Determination of similarities and
differences in the composition of individual molecules across the
geographical range of a species through mass profiling may also
represent a useful tool in chemotaxonomy [67] and for inferring [74]
or complementing [75] phylogeographical patterns generated by
genetic (mtDNA) approaches [76].
Proteotranscriptomics: proof checking the quality &
annotation accuracy of a species-specific contig
database using proteomics data
An interesting derivation of the next-generation snake venomics
protocol described above is the possibility of using the proteo-
mics data to check back the accuracy of the original transcrip-
tome assembly and translation (FIGURE 2). Thus, when the set of
(tryptic) peptides derived by proteolysis of an SDSPAGE-
separated protein band followed by data-independent MS/MS
acquisition (FIGURE 2; step 2) do not match a single species-specific
contig sequence (FIGURE 2; step 3), three distinct scenarios are possi-
ble: (i) some peptides are shared between different proteins each
of which also possess unique peptides (FIGURE 3A); (ii) the set of
tryptic fragments comprises different subsets of peptides that
map onto nonoverlapping regions of different contigs (FIGURE 3B);
and (iii) different set of tryptic peptides match sequences covered
by different reading frames of the same contig (FIGURE 3C). The first
possibility clearly points out to the presence of a mixture of
homologous proteins in the same electrophoretic band. Possibil-
ity ii would indicate the erroneous assembly of contigs B1 and
B2 (i.e., due to false overlap of short reads), whereas scenario iii
is seemingly due to a read sequencing error leading to a reading
frame shift. All these assembly errors can easily be detected and
corrected by carefully annotating the transcriptomic data based
on proteomic findings (FIGURE 3; steps 5 & 6). Although low
throughput and time-consuming, the resolving power of check-
ing back the contig database using proteomics data, critically relies
on implementing strategies to decomplex the venom proteome,
ideally achieving the level of individual toxins, prior to LCMS/
MS analysis. Digesting the venom proteins without any sample
fractionation step greatly suppresses any downstream opportunity
of bridging the gap between proteome and transcriptome.
Review Calvete
doi: 10.1586/14789450.2014.900447 Expert Rev. Proteomics
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Next-generation venomics Review
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The term proteogenomics has been coined [7780] to describe
the mapping of MS/MS-identified peptides to the specific
genome locus coding for these amino acid sequences. By anal-
ogy, the use of proteomics data to reassess the quality of the six
possible translation frames of a species-specific transcriptomics
database, and to match MS/MS-derived peptide sequences to
transcriptomics loci, thereby contributing to accurate assembly
and locus annotation, should be called proteotranscriptomics.
Antivenomics: a translational venomics platform
Snakebite envenoming is largely a neglected threat to public
health in tropical and subtropical regions of Africa, Asia, Latin
America and Oceania, affecting some of the worlds poorest rural
communities. An estimated 5.5 million people are bitten by
snakes each year, resulting in about 400,000 amputations, and
between 20,000 and 125,000 deaths; however, the true scale of
this disease of poverty may be much greater than these hospital-
based statistics [14,8184]. Despite these figures, which affect mainly
people involved in subsistence farming activities in vast regions of
the world, the burden of human suffering caused by snakebites
has only been added to the WHOs list of neglected condition in
April 2009 [85] and represents the most neglected of the neglected
tropical diseases [14,15]. The timely parenteral administration of an
appropriate antivenom remains for more than a century after the
development of the first serum antivenimeux by Calmette [8689],
and Phisalix and Bertrand [9092], the only currently effective treat-
ment for snakebite envenomings [93]. Poor access to health services
in these settings and, in some instances, a scarcity of antivenom
often leads to poor outcomes and considerable morbidity and
mortality [83,84].
2.0
1.0
0.0
2.0
1.0
0.0
2.0
1.0
0.0
17
8
9
1
2
3
4
5
7
8
9
DD

DD
PLA
2
s
10
12
13
14
15
11
16
17
19
Serine proteinases
CTL
PIII-SVMP
20
21
22
23
24
26
LAO
PIII-SVMPs
Cerastes cerastes (Morocco)

2
3
4
5
7
8
9
10
12
13
14
15
11
16
17
19
20
21
22
23
24
26

25
25 Peptides
(min)
0 50 100 150
A
B
C
Figure 3. Immunocapture efficacy of the F(ab)
2
antivenom CcMo_AV toward Moroccan Cerastes cerastes venom. Immunore-
activity of the Moroccan monospecific (C. cerastes) CcMo_AV toward proteins from the venom of C. cerastes from Morocco assessed by
second-generation antivenomics. (AC) Show, respectively, reversed-phase separations of 0.25 mg of the whole venom; the nonimmuno-
captured venom components; and the proteins retained and recovered from the F(ab)
2
antivenom affinity matrix. Column eluates were
monitored at 215 nm. Proteins within the immunocaptured and the flow-through fractions were identified by the venomics approach
described in the text and schematized in FIGURE 1, and quantified by comparing the areas of homologous peaks in the two fractions.
CTL: C-type lectin-like; DD: Dimeric disintegrin; LAO: L-amino acid oxidase; PIII-SVMP: Snake venom Zn
2+
-metalloproteinase of class III;
PLA
2
: Phospholipase A
2
.
Adapted from FIGURE 4 of [104]. Picture of Cerastes cerastes: author, H Krisp, Ulm, Germany; reproduced from [120] under Creative Commons
Attribution 3.0 Unported (CC BY 3.0).
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Because each snake possesses its own
venom with a distinctive mix of chemicals,
an antivenom developed for one snake may
not work against the venom of another spe-
cies. To complicate matters, the evolution
of venomous species and their venoms do
not always follow the same course, and the
identification of structural and functional
convergences and divergences among ven-
oms is often unpredictable by a phyloge-
netic hypothesis. Thus, a key issue in the
manufacture of antivenoms is the selection
of the venoms that are used in the immu-
nizing mixtures [14,94]. Owing to the large
intra- and interspecific variability in venom
composition [95], it is necessary to ensure
that the venoms used for immunization
generate antibodies effective in the neutrali-
zation of the most medically relevant toxi-
nological activities in a geographical setting
of deployment. The study of the preclinical
efficacy of antivenoms has experienced a
significant evolution in the last decades. Ini-
tially, the only test performed to ensure
such efficacy was the neutralization of
lethality, using animal models, most fre-
quently mice [95]. Although this test
remains as the gold standard for antivenom
in neutralizing efficacy [9698], the complex-
ity of the pathophysiology of snakebite
envenomings demands a more meticulous
analysis of efficacy, based on the testing of neutralization of other
toxic effects in addition to lethality. Hence, simple experimental
protocols have been implemented for the analysis of the neutraliza-
tion of hemorrhagic, myotoxic, coagulant, edema forming and
defibrinogenating activities, as well as for the neutralization of
enzymatic activities such as proteinase, PLA
2
and hyaluronidase
(reviewed in [96]). On the other hand, the design of venom mix-
tures should be based on a rigorous analysis of epidemiological,
clinical, proteomic, immunological and toxicological information.
In this sense, knowledge of the ontogenetic, individual and geo-
graphical intraspecific venom variability has applications for the
quality control of the reference venom pool for generating new
and more effective broad-ranging antidotes [94]. On the other
hand, assessing the cross-reactivity of a polyvalent antivenom
against venoms not included in the immunization mixture may
aid in expanding their range of clinical application [14,94]. The com-
bination of antivenomics and neutralization assays of toxic activi-
ties provides a powerful methodological platform to analyze, in
depth, the preclinical efficacy of antivenoms.
Confronting the problem of snakebite envenoming requires the
full potential of combining venomic tools and preclinical testing of
antivenom efficacy using functional neutralization assays [96]. In
this respect, our laboratory collaborates with the Global Snakebite
Initiative [14,15,94,99,100], an international collaborative project
aimed, among other goals, at developing new regional polyvalent
antivenoms for Asia and Africa. We have developed a proteomics-
centered tool, termed antivenomics, for aiding in the design of
optimized immunization venom mixtures and assessing the immu-
nological cross-reactivity of antivenoms toward homologous and
heterologous venoms [25,101103]. The most recent antivenomics
workflow, dubbed second-generation antivenomics, consists of a
step of immunoaffinity chromatography step on Sepharose-
coupled IgG, F(ab)
2
, Fab or Fab molecules, followed by the
proteomic analysis of the nonimmunocaptured (flow through frac-
tion) and the immunocaptured fractions (FIGURE 4) [25]. The percent-
age of a particular venom toxin retained on the immunoaffinity
column represents a measure of the preclinical potential of the
immobilized antivenom against that venom toxin present in the
sampled venom. This figure can be experimentally derived as 100-
([NR
i
/(R
i
+NR
i
)] x 100), where NR
i
and R
i
are the chro-
matographic peak areas of toxin i in the chromatogram of the
nonretained and the immunocaptured and eluted affinity column
fractions recovered in steps 2 and 3 of FIGURE 4A. Experiments in
which venoms are incubated with mock matrix and with matrix-
coupled preimmune antibodies, run in parallel to the immunoaf-
finity antivenomics analysis, serve as matrix and immunospecificity
controls, respectively (FIGURE 4B). Its quantitative character and
molecular resolution suggest the possibility for antivenomics of
Step 1 Step 2 Step 3
Antivenom
immobilization
Incubation of
venom and
antivenom
Elution
Non-retained
venom components
Immunocaptured
venom
components
Venom
Antivenom
NHS-sepharose
Venom
Non-specific IgG
NHS-sepharose
Controls
Matrix control Specificity control
Analysis of retained and non-retained
venom components
A B
Figure 4. Second-generation antivenomics. Scheme of the immunoaffinity capturing
antivenomics protocol developed by Pla et al. in 2012 [25]. (A) Whole venom is applied
to an immunoaffinity column (step 2) packed with antivenom antibodies immobilized
onto Sepharose beads (step 1). After eluting the nonretained venom components, the
column is thoroughly washed and the immunocaptured proteins eluted (step 3). (B)
Cartoon of the specificity controls of the immunoaffinity-based antivenomics protocol
schematized in (A). Mock Sepharose 4 Fast Flow matrix, (matrix control) or Sepharose
beads coated with preimmune IgG molecules (immunospecificity control), was incubated
with venom and developed in parallel to the immunoaffinity column. Qualitative and
quantitative analysis of the column eluates are illustrated in FIGURE 3.
NHS: N-hydroxysuccinimide.
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supplanting the use of immunoassays and western blots, the most
popular techniques for assessing the immunoreactivity of antibod-
ies. The capability of this simple knowledge-based analytical
method to formulate hypotheses as to how improved venom mix-
tures might be designed or redesigned for the manufacture of
improved therapeutic antivenoms has been documented in a num-
ber of investigations in recent years (FIGURE 3) (reviewed
in [16,103,104]). The following examples illustrate this point.
The ability of EchiTAb-Plus-ICP

antivenom to immunode-
plete and neutralize the venoms of African spitting cobras was also
assessed by antivenomics and neutralization tests [25,71,96]. The
antivenom neutralized the dermonecrotic and PLA
2
activities of
all African Naja venoms tested, whereas lethality was eliminated
in the venoms of Naja nigricollis, Naja mossambica and Naja pal-
lida, but not in those of Naja nubiae and Naja katiensis. Antive-
nomics analysis indicated that the impaired binding capability of
the antivenom to a type-1 a-neurotoxin, high abundant in N.
nubiae (12.6% of the total venom proteome), N. katiensis (4.4%)
and N. pallida (2.8%) venoms but absent in the N. nigricollis
venom used in the immunization mixture, appears to be responsi-
ble for the inability of EchiTAb-Plus-ICP antivenom to neutralize
lethality of of N. nubiae and N. katiensis venoms. The antivenom-
ics results suggest that N. nubiae venom should be included as
part of a improved immunization mixture, either in addition of
or substituting N. nigricollis venom.
Hypotheses are proposed explanations for observable phenom-
ena. The scientific method requires that one can test them, and
venomics- and antivenomics-based hypotheses are no exceptions.
In this regard, recent genus-wide venom proteomics analyses across
Lachesis revealed a high conservation of the overall composition of
Central and South American bushmaster venoms [105]. This find-
ing suggested that a monospecific antivenom generated against the
venom of any Lachesis species may exhibit paraspecific protection
against the toxic activities of all other venoms of congeneric spe-
cies [105]. This venomics-guided hypothesis was validated in a sub-
sequent study in which the paraspecificity of two antivenoms,
produced at Instituto Vital Brazil (Brazil) and Instituto Clodomiro
Picado (Costa Rica) using, respectively, Lachesis muta rhombeata
and L. stenophrys in the immunization mixtures, was assessed using
genus-wide comparative antivenomics [106]. Similarly, a combined
venomics and antivenomics investigation on the venoms of the
Taiwanese snakes, Protobothrops mucrosquamatus and Viridovipera
stejnegeri, showed that a bivalent antivenom generated against these
venoms was unable to neutralize the lethality induced by P.
mucrosquamatus [107]. This deficiency was associated by antivenom-
ics analysis with the antivenoms inability to recognize the crotoxin
B-like neurotoxic PLA
2
, trimucrotoxin, which accounts for 5.2%
of the total venom proteins of P. mucrosquamatus [107]. This find-
ing suggested that neutralization of the neurotoxic activity of tri-
mucrotoxin should neutralize the lethal activity of P.
mucrosquamatus venom. This hypothesis was verified by showing
that the anti-Crotalus durissus terrificus antivenom produced at
Instituto Butantan (Sao Paulo, Brazil) immunorecognized trimu-
crotoxin and neutralized the lethal activity of P. mucrosquamatus
venom [107]. The corollary of this research is that the bivalent
antivenom can be improved through enrichment of the immuniza-
tion mixture with a trimucrotoxin cross-immunoreacting PLA
2
molecule, such as the South American rattlesnake crotoxin.
Expert commentary
The increasing availability of comprehensive venomic, transcrip-
tomics and genomic databases for studying the interplay between
ophidian phylogeny, biogeography and organismal biology offers
an unprecedented opportunity to address fundamental questions
about the natural history of snakes and their venoms such as:
How did snakes evolve and what processes govern the generation
of venom convergences and divergences we see today? How do
snakes come to be adapted to their environment? Why do differ-
ent snakes use distinct venom formulations to subdue similar
prey? Like the story of the blind men who described differently
what an elephant looked like because each one touched a different
part of the elephants body, separately each omics approach reveals
only part of the system under study. Nothing in biology makes
sense except in the light of evolution is a 1973 essay by the evolu-
tionary biologist Theodosius Dobzhansky [108]. The central issue
of this essay criticizing antievolution creationism and espousing
theistic evolution, applies seemless to all fields of biology includ-
ing venomics. The fact that evolution occurs explains the interre-
latedness of the various facts of biology, and thus understanding
the molecular bases that have shaped the variation in venomous
snakes requires a joint analysis of the different omics data sets
within an evolutionary framework. The realization that conver-
gence in venom composition between the two marine snake line-
ages is driven by their parallel specialization for a fish-specific diet
explains the remarkable cross-reactivity between sea snake anti-
venom and sea krait venom [109]. On the other hand, the com-
monly occurring compositional variability of venoms from widely
distributed and highly adaptable terrestrial snake species is of par-
ticular medical concern, for allopatric venom variability represents
a source of diversity of the pathological effects of envenoming.
The environmental and molecular mechanisms generating this
diversity remain elusive, though a robust knowledge of the venom
arsenal within an accurate phylogeny and a detailed biogeographi-
cal pattern, along with the recognition of convergent and diver-
gent trends along the venoms evolutionary history, are of applied
importance for selecting the most suitable species and specimens
for the production of improved therapeutic antivenoms. The evo-
lutionary hypothesis represents the Rosetta Stone to bridge the
gap between the inventory of toxins and biology.
Five-year view
The recent sequencing of the King cobra [18] and Burmese
python [19] in conjunction with the publication of the genomes of
Anolis carolinensis [110,111] and the American alligator, Alligator mis-
sissippiensis, the gharial Gavialis gangeticus and the saltwater croco-
dile Crocodylus porosus [112], has barely begun to clarify aspects of
the biology of Archosauria (birds and crocodilians) and Squamata.
With over 9000 species among 61 families, squamate reptiles (liz-
ards and snakes) are one of the most diverse group of vertebrates.
Squamates offer outstanding model systems in ecology and
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evolution. As recent advances in sequencing technologies have
greatly reduced the time, cost and difficulty of generating novel
genomes, the next 5 years will undoubtedly witness the establish-
ment of significant genomic resources for more nonavian reptile
species including venomous and nonvenomous snakes. Compara-
tive genomics between birds, reptiles and mammals will provide
insights into over 300 million years evolution of amniotes [113]
including a key perspective on the implication of retroelements in
the genesis, genomic organization and evolution of venomous sys-
tems during the last 200 million years [2,114]. The explosion of
sequencing and annotation of complete nonavian genomes will
make the coming years exciting for evolutionary toxinologists.
Genetic variation is the driving force of evolution, and the nat-
ural selection acting on the organisms phenotype, the primary
mechanism for adaptation of the heritable variation that results in
fitness differences. Venom represents a trophic adaptive trait, cru-
cial to the foraging success of the snake. Bridging the gap between
genotype and phenotype requires both, a well-resolved phylog-
eny [115] and an understanding of the mechanisms controlling the
accelerated evolution of venom toxins [116], and the regulatory
components of the venom secretory system determining the pat-
tern of transcript abundance and translation. miRNA-mediated
post-transcriptional modulation of the venom transcriptome has
been hypothesized to contribute to venom evolvability without
large-scale alterations of the underlying gene expression machin-
ery [117]. The next 5 years will provide evidence to substantiate or
refute this hypothesis. This will require same species tissue-specific
transcriptomic and small RNA deep sequencing.
Deconstructing complex molecular phenotypes, such as snake
venoms, is within the reach of current omic technologies. Para-
doxically, the increased capabilities (and shorter duty cycle) of
mass spectrometers have led to high-throughput proteomics
increasingly functioning as a black box [118]. The increasing
availability of full-length genomic and transcriptomic databases
within the next 5 years will contribute to the explosion of venom
proteomics. Genus-wide venomics, antivenomics and bio-
geographical studies will reveal the chemical and immunological
space of venoms at different taxonomic levels. This information
is relevant for venomics to meet the challenge of snakebite enve-
noming in tropical and subtropical regions of Africa, Asia, Latin
America and Oceania. During the next years, next-generation
snake venomics research developed in close collaboration in the
Spanish Instituto de Biomedicina de Valencia and the Costa
Rican Instituto Clodomiro Picado [14,15,25,94,96,99,103107,119] will
continue to serve the interests of the Global Snakebite Initiative.
Hopefully, results of these multicentric collaborative studies will
contribute to improving the therapeutic management of snake-
bite and may ultimately reduce the current snakebite mortality
and improve the quality of life of thousands of men, women and
children in the worlds poorest communities.
Acknowledgements
Funding for the research described in this paper was provided by grants
BFU2010-17373 from the Ministerio de Ciencia e Innovacion (currently,
Ministerio de Economa y Competitividad), Madrid; PROMETEO/2010/
005 from the Generalitat Valenciana; CRUSA-CSIC (2009CR0021) and
CYTED project BIOTOX P211RT0412.
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial con-
flict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pending, or
royalties.
No writing assistance was utilized in the production of this manuscript.
Key issues
Though initially conceived as a technological platform, next-generation snake venomics also represents a conceptual framework for the
comprehensive analysis of venoms.
The availability of species-specific full-length venom gland transcript sequences as a reference database has greatly enhanced the efforts
of mass spectrometry-based venom proteomics, circumventing the need for de novo mass spectrometry sequencing.
Full proteome coverage at locus-specific resolution is within the reach of current omics technologies, but requires integrating hypothesis-
driven and technology-driven approaches. The need to decomplex the venom proteome represents an opportunity to quantitate the rel-
ative abundances of the different venom components.
An interesting derivation of next-generation snake venomics is the possibility of using the proteomics data to proof checking the
accuracy of the assembly and translation of the transcriptome database.
The recent publications of the King cobra and the Burmese python genomes provide insights into the biology and the evolution of
venom toxin genes at the genome structural level and represent the foundation of comparative snake genomics.
Developing the full potential of venom research requires the integration of data across the biological system within the frame of an
evolutionary hypothesis.
Bridging the gap between genotype and phenotype requires an understanding of the mechanisms controlling the accelerated evolution
of venom toxins and the regulatory components of the venom secretory system.
Genus-wide venomics, antivenomics and biogeographical studies will reveal the chemical and immunological space of venoms at
different taxonomic levels. This information is relevant for venomics to meet the challenge of snakebite envenomings.
Understanding evolutionary trends across venoms represents the Rosetta Stone for generating broad-ranging polyspecific antivenoms.
Next-generation venomics Review
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Papers of special note have been highlighted as:
of interest
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Selected applications of venomics and
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