Journal of Proteomics 135 (2016) 62–72

Contents lists available at ScienceDirect

Journal of Proteomics

journal homepage: www.elsevier.com/locate/jprot

Ecological venomics: How genomics, transcriptomics and proteomics can

shed new light on the ecology and evolution of venom
Kartik Sunagar a, David Morgenstern b,⁎, Adam M. Reitzel c, Yehu Moran a,⁎
a
Department of Ecology, Evolution and Behavior, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
b
Proteomics Resource Center, Langone Medical Center, New York University, New York, USA
c
Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA

a r t i c l e i n f o a b s t r a c t

Article history: Animal venom is a complex cocktail of bioactive chemicals that traditionally drew interest mostly from biochem-
Received 15 July 2015 ists and pharmacologists. However, in recent years the evolutionary and ecological importance of venom is real-
Received in revised form 2 September 2015 ized as this trait has direct and strong influence on interactions between species. Moreover, venom content can
Accepted 9 September 2015
be modulated by environmental factors. Like many other fields of biology, venom research has been revolution-
Available online 15 September 2015
ized in recent years by the introduction of systems biology approaches, i.e., genomics, transcriptomics and prote-
Keywords:
omics. The employment of these methods in venom research is known as ‘venomics’. In this review we describe
Venom the history and recent advancements of venomics and discuss how they are employed in studying venom in
Toxin general and in particular in the context of evolutionary ecology. We also discuss the pitfalls and challenges of
Genomics venomics and what the future may hold for this emerging scientific field.
Transcriptomics © 2015 Elsevier B.V. All rights reserved.
Proteomics
Venomics

1. Introduction venoms is of growing interest for the pharmacological and biotechno-

Venom is defined as a secretion, produced in a specialized gland or
cell of one animal that is actively delivered to the target animal through
the infliction of a wound. It is a complex mixture of toxin peptides, pro-
teins, salts and other chemicals employed mostly for prey capture and/
or for defense from predators and aggressors [1,2]. Further, venoms are
also employed by some animals such as leeches, ticks and vampire bats
to facilitate their specialized blood feeding habits [3,4]. As venoms are
characterized by an unusual diversity of components, and may contain
hundreds of toxin peptides they generate considerable interest among
evolutionary biologists and biochemists alike. The structural variability
of animal toxins is remarkable [5] and they exhibit a large array of bio-
logical activities [1]. Genes coding these peptides are hypothesized to
evolve under positive Darwinian selection due to their participation in
an evolutionary “arms race”, where the evolution of venom resistance
in prey and the invention of potent venom components in the secreting
animal exert reciprocal selection pressures [6–8]. On the other hand, it is
clear now that several protein families with non-venomous functions
are recurrently and independently recruited into the venoms of differ-
ent animal lineages [3], and that some venoms evolve mostly under pu-
rifying selection with only episodic positive selection [9]. The study of
⁎ Corresponding authors.
E-mail addresses: venomologist@gmail.com (D. Morgenstern),
yehu.moran@mail.huji.ac.il (Y. Moran).
logical communities as they are increasingly recognized as a rich source
for lead compounds that can drive forward the development of insecti-
cides and pharmaceutical drugs [10–12]. However, venom can also
serve as a model system where the relationship between genetic vari-
ability, protein biochemistry and interspecific interactions is more ame-
nable for elucidation and definition than in most other systems. After all,
an increase in the potency of venom can directly increase the fitness of
the predator secreting the venom and decrease the fitness of the prey or
vice versa, depending on which species is venomous. Thus, venom po-
tency can directly affect the strength of antagonistic interaction be-
tween the prey and the predator. Hence, evolutionary ecology can
greatly benefit from the study of venom. Unfortunately, in many cases
where much is known about venom composition we lack knowledge
regarding the ecology of the venomous animal and in many cases
where the ecology is well-understood little is known about venom com-
position. There are indications from snakes that the diet of a venomous
species might be closely-tied to its venom composition [13–15]. More-
over, it seems that cone snails employ different venom arsenals for
prey capture and defense [16] and that scorpions can control the com-
position of the mixture they inject via their sting [17]. It is plausible
that such tight relationships between interspecific interactions and
venom composition might be due to the high metabolic cost of venom
production, which makes potent venom advantageous, as less venom
is required for neutralizing prey or foe [18]. However, to better under-
stand such selective pressures we need a much better picture of the

http://dx.doi.org/10.1016/j.jprot.2015.09.015
1874-3919/© 2015 Elsevier B.V. All rights reserved.
 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
complete venom composition, its temporal dynamics and venom varia-
tion between individuals and populations as well as spatiotemporal var-
iations in diet and interspecific interactions. Filling these gaps with the
help of “omic” tools could help us understand the ecological factor and
evolutionary pressures that shaped the venom and also what role the
venom plays in the interspecific interactions of the venomous animal
and its ecological niche.
The term “venomics” was first used as a description for proteomic
study of snake venom composition [19,20], but in 2006 as a description
of an ambitious project aimed to provide a full picture of venom-related
biology by sequencing the full genomes, transcriptomes of venom
glands and venom protein contents of several venomous animal species
[21]. Since then it has also been used for describing studies of much
smaller scales that use several systems biology approaches for the com-
prehensive investigation of venom components, e.g., studies combining
shotgun mass spectrometry analysis of venom components with tran-
scriptome sequencing of a venom gland for charting an extensive list
of venom contents and getting a more complete picture on the proteo-
mic landscape of venoms and their biology [22,23]. The field of
venomics is growing fast but even with the current availability of new
high-throughput methods, the de novo sequencing of genomes,
transcriptomes and proteomes is far from an easy feat. In this perspec-
tive paper we will discuss the current state of knowledge, the pitfalls
and challenges awaiting for studies in this field and what are the future
directions that could help it grow in the context of evolutionary ecology.
2. Genomics and transcriptomics in venom research
2.1. Genomics of venomous animals
The DNA sequencing methods developed in parallel by Sanger and
Gilbert brought a true revolution to the field of biology [24,25]. Roughly
25 years later in a remarkable project by a large consortium and an in-
vestment of more than 3 billion US dollars and 10 years of work, the
full human genome was sequenced by applying the method developed
by Sanger [26]. As this method is costly and relatively slow, using it to
fully sequence a genome of several hundreds of millions of base pairs
(bp) would cost several millions of US dollars and would take several
years. However, a full-genome sequence of an organism is priceless for
biologists as it can give the full repertoire of genes, not only those that
are actively transcribed, but also the many additional genetic features
such as introns, intergenic regions and cis- and trans-transcriptional
regulatory elements that have pivotal roles in the control of gene ex-
pression and in the evolution and physiology of an organism [27]. Still,
the astounding costs have put a severe limitation on the ability to
sequence full animal genomes and very few genomes of venomous an-
imals have been sequenced to date. As an organism of pivotal agricultur-
al importance the European honeybee Apis mellifera was the first
Table 1
Sequenced genomes of venomous animals.
Organism Approximate genome size Sequencing platform
(million base pairs) (see Table 2 for details)
Arthropods
Acanthoscurria geniculata (tarantula) 6500 Illumina
Apis mellifera (honey bee) 262 Originally Sanger, later
454 and SOLiD
Mesobuthus martensii (scorpion) 1323 Illumina
Nasonia vitripennis (parasitoid wasp) 295 Sanger
Solenopsis invicta (fire ant) 352 Illumina and 454
Stegodyphus mimosarum (spider) 2550 Illumina
Cnidaria
Hydra magnipapillata 1050 Sanger
Nematostella vectensis (sea anemone) 357 Sanger
Vertebrates
Ophiophagus hannah (king cobra) 1590 Illumina
Ornithorhynchusanatinus(platypus) 1840 Sanger
63
venomous animal to be sequenced [28] (Table 1). However, it has an ex-
tremely streamlined venom with only a handful of components [29].
The next animal to have its genome sequenced was the starlet anemone
Nematostella vectensis that serves as an important model organism in
evolutionary developmental biology studies [30] (Table 1). This species
is a representative of the venomous phylum Cnidaria (sea anemones,
corals, jellyfish and hydroids), but the contents of its venom were un-
known before its genome was sequenced [31]. Thus, this species provid-
ed the first example for a genome-guided venom discovery [32,33].
Following Nematostella, the genomes of other cnidarians such as Hydra
magnipapillata and the reef-building coral Acropora digitifera were
sequenced as well [34,35] (Table 1). Another genome of a venomous an-
imal to be sequenced by the Sanger method is that of platypus
(Ornithorhynchus anatinus), which uses its venom for intraspecific ag-
gression among males during the mating season [36]. Lastly, the full ge-
nome of the parasitic wasp Nasonia vitripennis was sequenced by the
same method as well as partial genomic sequence of two other Nasonia
species [37]. The females of these tiny wasp species inject venom not
only for paralyzing host fly larvae, but also for manipulating the host's
gene regulation, possibly for making it more suitable for the needs of
their progeny [38]. The sequencing of the Nasonia genome provided
an important tool in the study of their venom that can be obtained
only in miniscule amounts, and hopefully provides an important step
in the direction of understanding how these complex genetic manipula-
tions are achieved [39].
The introduction of high throughput sequencing techniques, also
known as Next Generation Sequencing (NGS), in the last decade revolu-
tionized the field of genomics. Initially, pyrosequencing technology
platforms, such as those by 454 and then reversible terminator technol-
ogy platforms such as the ones from Illumina, made sequencing of mil-
lions of bps by a single run possible [40]. In recent years NGS outputs are
booming, for example, at the time of writing this manuscript (July
2015), the Illumina HiSeq 4000 sequencing system offered the ability
to sequence 1500 gigabases per run, i.e., to sequence about 12 human
genomes at a ×30 coverage in one run (Table 2). This second genomic
revolution has had a profound effect on all fields of biology as sequenc-
ing costs per bp are constantly decreasing. The NGS technologies en-
abled the sequencing of the genomes of the scorpion Mesobuthus
martensii[41] and the king cobra (Ophiophagus hannah) [42] (Table 1).
Sequencing of the full genomes of these two animals coupled to RNA-
Seq (the sequencing of expressed protein-coding genes via complemen-
tary DNA) revealed the nearly-full landscape of venom-encoding genes
in these animals. In addition, the genome sequences provided novel
insights into several evolutionary and zoological aspects related to
venom: from the ability of scorpions to resist their own venom to a pos-
sible answer to the enigmatic origin of venom glands in snakes [41,42].
NGS also enabled the sequencing of the first two spider genomes
[43] (Table 1). The vast majority of known spiders are venomous, and
Sequencing depth Scaffold N50 References for
(thousand base pairs) genome and venom
40× 47 [43]
6× (Sanger); 20× (SOLiD); Originally 359, [28,29,73]
4× (454) later 997
248× 223 [41,171]
6× 709 [37,39]
? 720 [46,47]
91× 480 [43]
8× 92.5 [34,172,173]
6.5× 470 [30,32,174]
? 226 [42]
6× 967 [36,175]
64 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72

Table 2
Major Next Generation Sequencing (NGS) platforms.

Platform Systems Sequencing Detection Run time Read length Reads per run Output
(in base pairs)

Illumina (previously Solexa) HiSeq 3000/HiSeq 4000 Synthesis Fluorescence b1–3.5 days ≥75% of bases Above Q30 5 billion 1.5 Tb
at 2 × 150 bp
Miseq Synthesis Fluorescence 56 h ≥70% of bases above Q30 25 million 13.2–15 Gb
at 2 × 300 bp
Life Technologies Ion torrent Synthesis Proton release 2h 200–400 4 million 1.5–2 Gb
(previously Invitrogen) Abi/solid Ligation Fluorescence 10 days 2 × 50 2.4 billion 240 Gb
Proton Synthesis Proton release 4h 125 60–80 million 8–10 Gb
Roche (previously 454) GS FLX+ Synthesis Pyrophosphate 23 h Up to 1000 1 million 700 Mb
GS Junior System Synthesis Pyrophosphate 10 h 700 0.1 million 40 Mb
Pacific Biosciences PacBio RS II Synthesis: one Fluorescence up to 240 min 10,000–15,000 0.8 million ~500 Mb–1 Gb
molecule at a time
Helicos Heliscope Synthesis: one Fluorescence 30 days 25,000–35,000 500 million 35 Gb
molecule at a time

Legend: bp: base pair; Mb: megabase pairs; Gb: gigabase pairs; Q: quality score.

the sequencing of the genomes of the African social velvet spider,
Stegodyphus mimosarum, and the Brazilian white-knee tarantula,
Acanthoscurria geniculata provided the first in-depth data regarding
venom genomics in spiders and the proteases that play an important
role in the maturation process of the small cysteine-rich peptides that
form the major groups of toxins in their venom.
NGS was recently applied in an unprecedented scale for the se-
quencing of the genomes of more than a dozen hymenopteran species
due to the great interest in their agricultural role, phenotypic plasticity
and in the evolution of eusociality [44,45]. It is likely that many of
these species are venomous, yet practically nothing is known about
their venom, with the notable exception of the fire ant, Solenopsis
invicta[46,47].
While genome sequences provide the full genetic data of a venom-
ous organism, they are not sufficient for determining the exact gene
boundaries and the cell and tissue expression dynamics and specificity
of genes. For addressing these questions transcriptomic and proteomic
approaches are required.
2.2. Venom transcriptomics
Transcriptomics show which transcripts are currently present in a
sample and hence enable the identification of genes that are transcrip-
tionally active (or at least been recently active) in a given animal, tissue
or cell. Further, transcriptomics also reveal untranslated regions (UTRs),
which can carry important regulatory roles and are many times difficult
to annotate accurately from genomic data. Early studies of venom
transcriptomes employed the preparation of complementary DNA
(cDNA) libraries by generating a double stranded cDNA and its cloning
into a plasmid vector, followed by sequencing by the Sanger method.
The resulting sequences are known as Expressed Sequence Tags
(ESTs). Despite its limited sequencing depth and relative cost inefficien-
cy compared with NGS approaches, this method is still occasionally
employed as it manages to give an initial insight into the venom content
of animals, as well as sheds light on the venom's physiological and bio-
chemical roles [48–51]. However, RNA-Seq methods are taking over the
whole field of transcriptomics, including venom transcriptomics, as
they are much more cost-effective due to their high-throughput nature
[52]. Transcriptomics are also used many times instead of genomics as
they cost only a small fraction of the latter due to the high intronic
and intergenic content of most eukaryotic genomes. Moreover, the
assembly of a transcriptome is a much simpler bioinformatic task
compared to genome assembly.
Transcriptomics enables the identification of various domains of the
transcripts including signal peptide, pro-peptide and the pre-pro
peptide domains that are rarely caught at the proteomic level due to
the fact that they are cleaved very quickly. Moreover, venom gland
transcriptomic investigations have provided novel insights into the
composition and evolution of venom in various poorly studied venom-
ous animals, such as sea anemones, polychaete worms, remipede crus-
taceans, ticks and platypus [53–57]. The transcriptomic approach is
especially important when proteomics are not feasible due to limited
venom accessibility. As reviewed by von Reumont et al. in 2014 [58],
there are multiple phyla whose venom is inaccessible for proteomic in-
vestigation due to the very limited size or number of venom glands or
the inability to extract venom from them. In such cases the fact that
transcriptomics incorporate PCR-based amplification steps is invalu-
able, even if it comes at the price of less accurate quantification due to
amplification biases.
Venom-gland transcriptomics involve the sequencing of mRNAs ex-
tracted from the venom gland to reveal a snapshot of the gland's tran-
scriptome at a given time point, which reflects the recent expression
of genes encoding venom components. This strategy not only permits
the identification of novel bioactive components in venom but also in
understanding how each toxin family varies between species or within
various populations of the same species. It can further shed light into
shifts in toxin expression profiles across developmental stages. Indeed,
ontogenic or developmental stage associated variation in venom has
also been studied by sequencing the venom gland transcriptomes of
various developmental-stages in snakes [59–61]. Application of integra-
tive omics technologies in the central American rattlesnake (Crotalus
simus simus) revealed an ontogenic shift in venom expression [59]. Pro-
teomic examinations (approaches discussed below) in this study
identified ontogenetic shifts in the venom that involved a gradual re-
duction in the expression of certain toxins, such as serine proteinases
and PLA2s. When these snakes reached an age of 9 to 18 months, an in-
creased secretion of PI and PIII metalloproteinases was observed. Deep
sequencing of small RNAs in the venom glands of various developmen-
tal stages identified several microRNAs (miRNAs) that were found to be
complementary to the transcripts of various venom protein encoding
mRNAs. Hence, the authors speculated that miRNAs may participate in
regulating the venom gland expression profiles of C. s. simus, and thus
result in ontogenetic-shifts in venom-composition. However, since
miRNAs are mainly known to fine-tune expression profiles in most
other animals and they affect gene expression and protein output by
less than 2-fold [62,63], the actual contribution of miRNAs in introduc-
ing variation in snake venom composition remains to be experimentally
validated.
Transcriptomics can also permit the understanding of how various
factors influence the composition of venoms, and ultimately the ecology
of venomous animals. Specifically, there are strong indications from
several studies in venomics that various ecological and environmental
factors, such as diet [15,64], gender [64,65], geographical distribution
[15,66], and temperature and UV radiation [67] may influence venom
composition in animals. Variability in venom is not only observed across
species, but within species as well, where different populations utilize
 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
different venom proteins for prey incapacitation [14,68]. An intriguing
question is what part of the variability in venom measured in individ-
uals from the same species is derived from genetic variation and how
much is due to plasticity of gene expression in response to environmen-
tal experience. Like any quantitative character, the particular phenotype
(here, venom composition) potentially reflects both: certain popula-
tions may have locally adapted venom composition resulting from ge-
netic adaptation over numerous generations (i.e., genotypic variation)
and individuals within each population may have the molecular mech-
anism to modulate venom composition based on within lifetime experi-
ence (i.e., environmental variation). Moreover, individuals within a
species may differ in the relative extent of modulations to their venom.
Venom gland transcriptomics of various populations of a species can
help in revealing such differences in venom compositions. For example,
a combined proteomic and transcriptomic investigation of the venom
glands of various populations of Crotalus oreganus helleri (the Southern
Pacific Rattlesnake), one of the most medically significant snakes in all
of North America, revealed remarkably different venom compositions,
despite relatively short geographical distribution of the examined pop-
ulations [69]. While hemorrhagic and tissue destroying toxins, such as
the snake venom metalloproteinases (SVMPs) were abundantly secret-
ed by the Catalina Island and Phelan populations, the Loma Linda and
the Idyllwild populations secreted SVMPs in moderate and trace
amounts, respectively. Moreover, the presynaptic neurotoxic PLA2 com-
plex, previously only identified in the Mohave rattlesnake (Crotalus
scutulatus) and the neotropical rattlesnake (Crotalus durissus terrificus)
were identified in the Idyllwild population of the San Jacinto Mountains.
Thus, transcriptomic and proteomic examinations revealed why
treating envenomations by this species has been a clinician's nightmare.
Venom transcriptome sequencing and evolutionary assessments also
identified variations in the molecular evolutionary rates of various
toxin families in distinct populations [69].
To fully discern the effects of genotype and environment in the com-
position of venom, the most straightforward approach would be to
compare the venom of genetically identical individuals exposed to a
range of environmental conditions. The use of clonal individuals like
those available for cnidarians or male hymenopterans removes genetic
variation from any measured transcriptomic and/or proteomic response
in the phenotype. Then, by comparing this response in multiple clonal
lines from a single species, one could quantify the genotype vs. environ-
ment contribution to the phenotypic variation. To our knowledge, this
experimental design has yet to be employed in the study of venomics.
2.3. Challenges for venom genomics and transcriptomics: the devil is in the
assembly and annotation
While NGS methods provide extraordinary amounts of data they
also provide new challenges: most of these methods provide reads
length of about paired-end 150–300 bp, whereas the Sanger method
can provide reads of about 700–1000 bp (Table 2). This has dramatic
effect on the ability to assemble transcripts and genes, as demonstrated
by the considerably shorter scaffolds obtained in many of the
genome projects that employed NGS techniques rather than Sanger
(Table 1). Moreover, toxin genes are especially prone to mis-assembly
as a single genome may contain numerous copies that may differ from
one another by only a handful of nucleotides [33]. This is extremely
problematic as native single amino variations in a toxin can result in
dramatically different pharmacological activities [70]. Assembly of
venom gland transcriptomes was shown to be especially prone to this
typical sequence variation as most common transcriptome assemblers
such as Trinity [71] are designed to collapse such “minor” differences
into a single transcript as slight variation in most other systems is con-
sidered to be the result of either technical sequencing artifacts or allelic
variation that is unimportant in the context of generating a reference
transcriptome. Hence, it is important to develop assembly tools such
as VTBuilder [72], which is specifically built to capture those differences
65
and assemble them into distinct transcripts. However, setting the
threshold in a way that will ideally balance between real variation and
erroneous reads remains a challenge.
A possible solution for assembling longer genomes is the combina-
tion of Sanger sequencing and NGS: the Sanger sequencing provides
longer reads for bridging, but at a much higher cost per bp, whereas
the NGS provides numerous short reads at a much cheaper price. The
honeybee genome was recently re-assembled from a combination of
reads from various sequencing methods, resulting in much longer and
informative genomic scaffolds [73] (Table 1). Another solution is the
production of several distinct NGS libraries with vastly different insert
sizes. This approach produced a well-assembled spider genome that
was sequenced exclusively by the Illumina HiSeq technology [43]
(Table 2). Lastly, some of the NGS platforms like Heliscope, PacBio
(Table 2) or MinION [74] can provide extremely long reads that can
prove very useful when combined with short read data from other plat-
forms [75,76]. These platforms also allow to sequence single molecules,
enabling sequencing of full transcripts up to 10 kb in length [77], a qual-
ity that may prove extremely useful for documenting the true variability
of toxin-encoding transcripts, including specific alleles with all linked
polymorphisms.
Another major challenge in genomics of eukaryotes is annotating full
genes, from their transcription start site (TSS) to the last nucleotide,
while encompassing all exons, including the alternative ones in splice
variants. In the past a major part of gene annotation was based on pre-
diction algorithms such as Genscan that used mostly common patterns
in genomic sequences that typify exons-introns boundaries [78]. The
combination of transcriptomics and genomics for gene prediction and
annotation leads to a significant improvement in accuracy, and there
is no doubt that for this task NGS data is priceless, as a single sequencing
run can cover even the rarest splice variants. However, how to combine
all these data together in order to map full genes and to avoid artifacts is
not a simple task and various softwares that employ distinct approaches
to reach this aim are available [79]. Beyond the annotation of physical
gene structure, annotating function is of paramount importance.
Genomes of model organisms such as yeast, flies, nematodes and mice
provide the gold-standard for functional annotation, as genes in these
organisms were studied at extreme depth, the function of most of
their genes (but not all) is presently known. Further, the large research
communities using these organisms make the functional annotations
available in well-maintained public databases [80–82]. Functional an-
notation of genes in non-model organisms, such as venomous animals,
is based mostly on the homology to annotated genes of model
organisms. However, the problem is that toxic/venomous function is
non-existent in classic model organisms and as many toxin genes
were recruited from genes of non-venomous function [3] they might
be annotated with the wrong function. The use of manually annotated
databases that specialize in toxins can help in the process of annotating
novel toxin genes according to sequence homology [83,84]. However,
another problem arises due to the homology of toxins and the original
physiological proteins they were recruited from. Differentiating the
two apart can be difficult. An example for such difficulties can be
found in the work of Whittington et al. [57] where 454 as well as
Illumina NGS platforms were used for sequencing the venom gland of
platypus, and the data was compared to older Sanger data for tran-
scripts of this animal's bill, brain, liver, spleen, and testis. The idea was
to use the data from the non-venom-related tissues to exclude physio-
logical genes that are not toxins. The problem is that venom genes, as
least in early evolutionary stages after recruitment might be expressed
in both venom glands and other tissues in parallel and setting an appro-
priate cutoff is challenging. Moreover, expression quantification in the
platypus study was especially problematic as the Sanger data was limit-
ed for the non-venomous tissues. The authors arbitrarily decided that
only genes which are expressed in three or more non-venomous tissues
are not toxin-encoding genes. The problematic aspects of this study
were criticized by Terrat and Ducancel [85] and in this correspondence
66 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
the authors discussed what possible criteria can or cannot be used to an-
notate a toxin based on nucleotide data and homology. Another study
[86], which encountered similar problems, attempted to test the com-
mon origin of venom in Toxicofera — a hypothetical clade that includes
venomous snakes and lizards [87,88]. While the data generated in this
study was interesting and highlighted the importance of quantitative
comparison of venom proteins in the venom gland and physiological
tissues, it contained questionable interpretations and conclusions. The
authors arbitrarily classified a protein as highly secreted only if it repre-
sents the top 25% of all transcripts in at least two out of four examined
venom gland tissues. Any protein that did not represent the top 25% of
the transcript was considered to be playing a homeostatic function.
Thus, the authors likely ignored venom components that may be secret-
ed in lower amounts but could be potent enough none-the-less. More-
over, the potency of venom could be increased when components
function in synergy, as was shown for several scorpion toxins [89].
Since venom is considered as energetically expensive to secrete [18],
an extremely toxic component could be secreted in limited amounts
in the venom gland. Hence, it becomes essential to employ toxicity
assays that can reveal whether a particular component secreted in
what seems to be an insignificant amount actually participates in the
envenoming process or not.
Another challenge with annotating transcripts and genes as toxin-
encoding is the fact that the sequenced animal may have novel toxin
types that are non-homologous to previously studied toxins. Sequenc-
ing by low coverage (36,864 ESTs) the transcriptome of nematocytes
(the cnidarian stinging cells) of Hydra revealed that at least 15% of the
clusters did not have any significant homology to a previously known
protein [90]. Along the same lines, out of 20 proteins which are released
from the nematocyst (stinging capsule) of Nematostella during dis-
charge (stinging), only six had clear homologs in other organisms
[32]. Hence, homology-based annotation of toxins from transcriptomic
and genomic data can be insufficient for giving a full picture of the
venom arsenal of a venomous animal and proteomics are many times
essential in order to determine what proteins are actually secreted
from a venom gland or a venom producing cell and can be considered
as reliable toxin candidates.
3. Venom proteomics
The availability of genetic information allows for the identification of
the footprint of evolutionary processes through the history of the
species in question, either in comparison to other species or within
the species population. However, genetic data provides only a part of
the picture; genomic and transcriptomic data cannot always provide
accurate quantitative data [91–93] nor can it provide insight into post-
translational modifications [81,94,95], which may be important for the
toxin's functionality and therefore, for understanding its evolution and
ecological role.
3.1. Venom proteomics in the pre-genomic era
Modern proteomics rely heavily on the application of mass spec-
trometry to analyze complex proteinaceous mixtures. The ability to con-
vert the acquired spectra into protein identification relies almost
exclusively on the availability of a genomic database [96,97]. As a result,
venom proteomics flourished in recent years due to the affordability of
NGS. However, evolutionary venom proteomics have relied on the
availability of sequence information as early as the 1960s [98] and the
main challenge in this era resulted from the difficulty of obtaining
sequence information en-masse (through de-novo sequencing of indi-
vidually isolated proteins). These difficulties resulted in two distinct ap-
proaches of early venom proteomics — either through sequence based
phylogenetic analyses [99] or through a venom-centric description of
their composition by low-resolution profiling through electrophoretic
[98,100–103] and chromatographic [104–107] patterns. The latter
approach provided a unique and exciting opportunity for comparative
analyses of whole venoms, rather than concentrating on specific toxin
families. Indeed, this approach continues to be popular, and with the
improved resolution of modern chromatography and 2D-
electrophoresis a greater degree of detail can be achieved [108–111].
Additionally, these two analytical methods could be combined with
zymography or functional assays of chromatographic fractions to
allow the functional characterization of unique gel spots or fractions.
These approaches enabled the onset of fascinating studies tying var-
ious factors to variability in venom composition — the canonical work
by Daltry et al. [15], tied dietary differences in geographic populations
of a pitviper species with geographic variations, sex-related venom
variability [109,112], or a basic population proteomics analysis [113].
However, this approach provides a lower resolution overview of
venom compared to the level achieved by sequence-based approaches
(genomic or proteomic); chromatographic peaks or gel spots often
contain multiple proteins, even under orthogonal separations, while
multiple peaks and gel spots may contain various proteoforms [114]
and user-induced artifacts (oxidations, carbamylations and degrada-
tion). Moreover, successful experiments require large quantities
of starting material, biasing early studies to organisms producing
such quantities (i.e., snakes). Thus, qualitative and quantitative analyses
of sequence variants and their phylogenetic relationships require
higher sensitivity and considerably higher resolution, such as mass
spectrometry.
3.2. Venomics and mass spectrometry
The pivotal work of Tanaka et al. demonstrated the ability to identify
proteins using mass spectrometry [115]. It provided the unique ability
to improve the resolution of protein analysis down to unique
proteoforms (see definition at [116]), with mass spectrometry analyses
of venoms coming soon afterwards [117,118]. Mass spectrometry
showed superiority in sensitivity and resolution compared to chroma-
tography and electrophoresis, while providing identifiable sequence in-
formation for distinct precursors. In combination with fractionation, the
separation and identification of individual venom components became
feasible by mass spectrometry as it allows the observation of individual
components in venom. Unfortunately, limitations of instrument resolu-
tion favored the study of venoms rich in small peptides such as those
from scorpions and spiders [65,119,120]. In contrast, the sequencing
of the toxins in question remained a very low throughput process, with-
out the availability of a genomic database. To this day, most software an-
alyzing mass spectral data require a sequence database to compare the
spectra against (see [121] for review on interpretation of data mass
spectrometry). Lack of such databases due to high costs of genomic
sequencing in the early days of venom mass spectrometry made it ex-
tremely difficult to accurately identify individual protein toxins. The
availability of genomic databases provided the means to rapidly identify
protein groups. In most cases, these analyses are performed “Bottom-
Up” (Fig. 1): the proteome is either digested unfractionated (“shotgun”)
or following fractionation by chromatographic or electrophoretic
means. The digests are then analyzed by the mass spectrometer and
the data generated are then matched against the genomic database.
This approach rarely provides the identification of the full toxin se-
quences, even with a fully accurate genomic database. Still, it is suffi-
cient to match MS data to associate chromatographic peaks or gel
spots to identify a family of toxins, providing the opportunity to give a
functional context to the variability observed in earlier studies. As a re-
sult a flood of studies looking at intra- [122,123] and inter- [19,124–
127] specific variations in venom composition from various phyla, as
well as ontogenic [122,128], sex [129], geography [122,130,131] and
diet [132] based variations, were conducted.
Later studies have demonstrated an additional utility of combining
proteomic and genomic analyses: while traditionally proteomic analy-
ses followed the process of genomic data generation and library
 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72 67

assembly, combining proteomics and genomics in the process of ge-
nome/transcriptome assembly, or proteogenomics, allows the identifi-
cation and annotation of novel unidentified proteins [133,134], as well
as improved genomic sequence assembly [135].
3.3. Current achievements and ongoing challenges in venom proteomics:
top-down proteomics
High throughput proteomics have flourished in recent years as a re-
sult of the availability of genomic databases and search software, yet the
determination of individual intact proteoforms remains a challenge. Ad-
vances in instrumentation and data analysis in the last decade has slow-
ly brought forth the ability to characterize individual intact proteins
(i.e., Top-Down; Fig. 1) at a considerably faster pace than ever before,
and at a higher throughput (see for review [136]). This capability is
especially appealing for venom research, as even very slight variations
in sequence or post-translational modifications (PTMs) may result in
significant changes in activity (see example for the effect of point muta-
tions in synthetic [137] and native [70] toxins), making their identifica-
tion paramount. Indeed, Top-Down venom proteomic analyses were
performed recently on peptidic venoms [138,139] as well as small-
protein venom [140]. Unfortunately, these experimental achievements
highlighted the inadequacy of the downstream data analysis; proteo-
mics search engines require utmost sequence accuracy, as sequence
variants and unknown PTMs (compared to the database) will result in
false negative identification of the protein in question. The current solu-
tion in most cases is improving the database through the production of
extensive variant libraries or individual genomic libraries. This solution
is typically not feasible for non-model organisms in general and venom-
ous animals in particular, due to the associated cost and effort required
in production and assembly of genomic and transcriptomic libraries for
every organism in question. The current viable option is the use of toler-
ant database search engines, such as Byonic [141] and ProSight [142],
that allow the identification of proteins that are very similar to se-
quences in the database, yet not identical. This type of analysis provides
identification (and maybe localization) of the similar protein, yet it does
not provide an alternative identification. It remains to the user to iden-
tify the correct sequence of the protein in question. This requirement
slows down the analysis considerably, yet it is immensely quicker
than manual de-novo sequencing.
What about automated de-novo sequencing? There are currently
several de-novo solutions in the market, either free [143–145] or com-
mercial [146]. These methods may well be effective for simple spectra,
derived from short peptides, yet they are still inadequate for the analysis
of spectra derived from larger peptides and proteins. This is not to say
that de-novo sequencing has no future in Top-Down proteomics;
though still in development, multiple publications have presented
some promise for Top-Down proteomics, either through a combination
of Bottom-Up and Top-Down data [147,148] or through a constrained
approach [149] that requires a sequence “anchor” from which the de-
novo algorithm works.
Despite its limitations Top-Down proteomics, either in its more
“primitive form” or the modern one, provided an immensely important
finding about venom biology: the understanding that venom produc-
tion and secretion is not monolithic and uniform, but rather a complex
mixture whose composition is susceptible to biochemical and behavior-
al modulation. Studies have hinted of this phenomenon in scorpions
[17,103], snake [150] and cone snails [16,123,151]. It is still unclear
what evolutionary processes drive venom modulation, but it provides
the crucial context to evolutionary signals identified from the nucleo-
tide data.
3.4. Current achievements and ongoing challenges in venom proteomics: in
situ venom proteomics
Another significant challenge facing the field of venomics is venom
accessibility. Venom studies have concentrated so far on organisms
whose venom is easily accessible — first snakes and scorpions, followed
by spiders, cone snails and then a whole plethora of venomous organ-
isms, from vampire bats [4] to Arctic cephalopods [152]. Unfortunately,
in many of these cases the “venom” sample is really a whole tissue ly-
sate containing the venom gland, leading to a contamination of the

Fig. 1. Typical workflows for “Bottom up” and “Top down” proteomic experiments. Typical proteomics analysis begins with protein extract, which is in our case milked or extracted venom.
The sample undergoes the reduction of disulfide bonds and cysteine alkylation to prevent the reformation of disulfide bonds to enable fragmentation in the mass spectrometer. Under a
Bottom-up approach the venom is enzymatically or chemically digested, generating short sequence fragments which are then acquired by the mass spectrometer. These short reads are
then searched against a known protein database. Identified peptides are used to reconstruct. While this method is more suitable for most mass spectrometers, as well as search engines, it
suffers from drawbacks such as incomplete coverage (illustrated by the missing green lines) and inability to reconstruct the correct proteoform state of protein (colored balls). In contrast,
in top-down proteomics, the proteins are analyzed intact, skipping sample processing and retaining any information related to the proteoform state of the sequence. However, top-down
acquisition of data requires specialized instruments, software and higher skill in experimental design. It is also less adequate for analysis of large proteins or proteins of low solubility.
68 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
venom with cellular proteins. Still, one may consider such contamina-
tion as the lesser evil, as most venoms are completely inaccessible
[58]. Even in the case of the extensively studies spider venoms, one
can find many clades of spiders too small to analyze their venom
successfully — enough to prevent gaining a full picture of the Araneae
venomic landscape. Quantitatively, those inaccessible species include
N40,000 species of spiders, ~ 10,000 species of pseudoscorpions,
N40,000 species of Acari and thousands of other species across the
tree of life.
In-situ mass spectrometry, in the form of Imaging Mass Spectrome-
try (IMS), may circumvent this problem; IMS provides a unique oppor-
tunity to provide information about the arrangement of proteins and
small molecules in the tissue, highlighting spatial specificity of mole-
cules that will be lost otherwise in tissue extraction (for reviews see
[153,154]). However, the basic capability of this technique in analyzing
proteins straight off tissue provides in immensely important tool for
venom researchers to study. Both Bottom-Up [155] and Top-Down
[156] proteomics have been demonstrated in the context of imaging
mass spectrometry, and while it is not a mature technology as of yet,
current developments in instrumentation and data analysis suggest
that we may be able to perform routine off-tissue proteomics within
the next decade.
While in-situ sequencing may be still in the future, several exciting
studies have been published utilizing IMS to provide information of
the arrangement of toxins in the secretory tissue. Undheim et al. have
presented convincing evidence for spatial differentiation of toxin pro-
duction in the venom glands of centipedes due to ecological pressures
[127,157], and such differentiation was also detected in cone snail
venoms due to the differentiation of the venom into scenario specific
venoms that are stored in distinct and separated compartment [16].
3.5. Current achievements and ongoing challenges in venom proteomics:
quantitative venom proteomics
One of the biggest advantages of protein-based analyses is their abil-
ity to convey quantitative information of various toxins in the venom
and between individual venoms. Early studies have used indirect ap-
proaches, particularly ELISA [158–160], enzymatic studies [158–162],
chromatography [163] and electrophoresis [164,165], providing quanti-
tative information without the need for sequence data. However, the
quantitative data available with these methods is of low resolution,
and cannot distinguish between individual proteins with similar activi-
ties (although 2D zymography may come very close to it). More impor-
tantly, these methods disconnect the data derived from proteins, from
any evolutionary information derived from genomic or transcriptomic
data. To achieve this connection, mass spectrometry-based proteomics
is a key technology. Quantitative mass spectrometry has developed im-
mensely in the recent decades (see reviews [166,167]), dividing into
two main experimental approaches — relative and absolute quantita-
tion. Relative quantification is the most common approach as it is the
easiest to perform. It compares the abundance of the same peptide or
protein based on the spectral data of the precursor itself (MS1), its
fragments (MS2) or a reporter ion (chemical labeling). In most cases,
protein quantitation is performed on a digest of the sample. Unfortu-
nately, Bottom-Up quantitative proteomics suffer from all of the prob-
lems listed in the above section dealing with Top-Down proteomics as
many individual toxins carry a single peptide that is unique to them
and distinguishes them from other variants. The reliance on a single
peptide for quantitation is highly unfavorable in quantitative proteo-
mics and should be avoided. As a result, neither label-free [43] nor
chemical labeling [128,168] took the same importance in venomics as
in general proteomics.
A possible solution would be to adopt a Top-Down approach for
quantitation, absolving the need to identify a specific digested peptide
for the quantitation of a specific toxin. Unfortunately, the technical dif-
ficulties in the analysis of proteins (N20 kDa) limit Top-Down
quantitation using mass spectrometry to peptidic venoms. Limited
Top-Down quantitation of MS1 spectra of venoms has been published
previously using label free quantitation [16,123]. Quantitation of
venoms composed of large proteins is feasible using a veteran method
— 2D gel electrophoresis, using a DIGE (Differential Gel Electrophore-
sis). While this method may not match the resolution of mass spectrom-
etry, the degree of separation for most venoms is probably sufficient.
Protein identification can be performed either from a reference gel or
from the DIGE gel itself using Bottom-up approach afterwards. Curious-
ly, despite the obvious advantages of DIGE for venom research (espe-
cially snake venom research), DIGE was mainly used to identify
qualitative differences between distant venoms, rather than being uti-
lized for fulfilling its quantitative potential [169,170]. Follow-up analy-
sis with Bottom-Up proteomics of individual gel spots provides the
protein identification to match to the quantitative information.
While relative quantitative venomics is gradually taking off, this is
not the case for absolute quantitation. The difficulties associated with
absolute quantitation, requiring known amounts of non-labeled or
(preferentially) isotopically labeled peptide for each precursor in ques-
tion, make this technique largely inaccessible as comparative analyses
between different toxins within a single venom or between venoms
will require large monetary investment in the production of labeled
peptides or large-scale purification of the individual toxins in question.
As the quantity of material tends to be a limiting factor in many
venomics studies, it is difficult to envision the incorporation of absolute
quantitation as a staple method for venom research. Instead, lower
accuracy methods such as densitometry, may be the most realistic
option for future quantitative analyses.
4. Concluding remarks
Venomous animals are usually highly dependent on their venoms
for survival, presumably placing this trait under extreme levels of selec-
tion. Further, venom plays an important part in the interspecific interac-
tions of the animals producing it and in shaping the ecological role of a
species. There is little doubt that omic techniques are providing power-
ful instruments for understanding the evolutionary ecology of venom,
yet like any other scientific method they should be utilized properly
and their results should be interpreted with caution.
The field of venomics is undoubtedly benefiting from the current
advances in proteomics, transcriptomics and genomics. The unique bio-
chemical nature of venom; i.e., the fact that it contains a large array of
sequence variants whose differences are often significant for their activ-
ity, presents a special challenge for omics analyses. Still, it appears that
the field is yet to make full use of the newest technologies available.
We expect that the promiscuity of high resolution and high throughput
instrumentation, along with improvements in data-analysis technolo-
gies will lead to improved accessibility of these methods to venom
researchers. Further, we believe that this improved availability of the
newest methods will enable research in the near future that will provide
new dimensions into the evolution and ecology of venom.
Transparency document
The Transparency document associated with this article can be
found in the online version.
Acknowledgments
KS was supported by a Marie Skłodowska-Curie Individual Fellow-
ship (654294). The work in the research group of YM is supported by
the Israel Science Foundation grant no. 691/14. Work on cnidarian
venom ecology in the labs of YM and AMR is supported by the Binational
Science Foundation grant no. 2013119.
 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
References
[1] N.R. Casewell, W. Wuster, F.J. Vonk, R.A. Harrison, B.G. Fry, Complex cocktails: the
evolutionary novelty of venoms, Trends Ecol. Evol. 28 (2013) 219–229.
[2] D. Mebs, Venomous and poisonous animals: ahandbook for biologists, toxicologists
and toxinologists, physicians and pharmacists, Medpharm, 2002.
[3] B.G. Fry, K. Roelants, D.E. Champagne, H. Scheib, J.D. Tyndall, G.F. King, et al., The
toxicogenomic multiverse: convergent recruitment of proteins into animal
venoms, Annu. Rev. Genomics Hum. Genet. 10 (2009) 483–511.
[4] D.H.W. Low, K. Sunagar, E.A.B. Undheim, S.A. Ali, A.C. Alagon, T. Ruder, et al.,
Dracula's children: molecular evolution of vampire bat venom, J. Proteome 89
(2013) 95–111.
[5] S. Mouhat, B. Jouirou, A. Mosbah, M. De Waard, J.M. Sabatier, Diversity of folds in
animal toxins acting on ion channels, Biochem. J. 378 (2004) 717–726.
[6] T.F. Duda Jr., S.R. Palumbi, Molecular genetics of ecological diversification: duplica-
tion and rapid evolution of toxin genes of the venomous gastropod conus, Proc.
Natl. Acad. Sci. U. S. A. 96 (1999) 6820–6823.
[7] K. Nakashima, T. Ogawa, N. Oda, M. Hattori, Y. Sakaki, H. Kihara, et al., Accelerated
evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes,
Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 5964–5968.
[8] K. Sunagar, W.E. Johnson, S.J. O'Brien, V. Vasconcelos, A. Antunes, Evolution of
CRISPs associated with toxicoferan-reptilian venom and mammalian reproduction,
Mol. Biol. Evol. 29 (2012) 1807–1822.
[9] M. Jouiaei, K. Sunagar, A. Federman Gross, H. Scheib, P.F. Alewood, Y. Moran, et al.,
Evolution of an ancient venom: recognition of a novel family of cnidarian toxins
and the common evolutionary origin of sodium and potassium neurotoxins in
sea anemone, Mol. Biol. Evol. 32 (2015) 1598–1610.
[10] J.K. Klint, S. Senff, D.B. Rupasinghe, S.Y. Er, V. Herzig, G.M. Nicholson, et al., Spider-
venom peptides that target voltage-gated sodium channels: pharmacological tools
and potential therapeutic leads, Toxicon 60 (2012) 478–491.
[11] J.J. Smith, V. Herzig, G.F. King, P.F. Alewood, The Insecticidal Potential of Venom
Peptides, Cell. Mol. Life Sci. (2013).
[12] I. Vetter, R.J. Lewis, Therapeutic potential of cone snail venom peptides
(conopeptides), Curr. Top. Med. Chem. 12 (2012) 1546–1552.
[13] A. Barlow, C.E. Pook, R.A. Harrison, W. Wuster, Coevolution of diet and prey-
specific venom activity supports the role of selection in snake venom evolution,
Proc. Biol. Sci./R. Soc. 276 (2009) 2443–2449.
[14] T. Chijiwa, M. Deshimaru, I. Nobuhisa, M. Nakai, T. Ogawa, N. Oda, et al., Regional
evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus
flavoviridis snakes in the southwestern islands of Japan, Biochem. J. 347 (2000)
491–499.
[15] J.C. Daltry, W. Wuster, R.S. Thorpe, Diet and snake venom evolution, Nature 379
(1996) 537–540.
[16] S. Dutertre, A.H. Jin, I. Vetter, B. Hamilton, K. Sunagar, V. Lavergne, et al., Evolution
of separate predation- and defence-evoked venoms in carnivorous cone snails, Nat.
Commun. 5 (2014) 3521.
[17] B. Inceoglu, J. Lango, J. Jing, L. Chen, F. Doymaz, I.N. Pessah, et al., One scorpion, two
venoms: prevenom of Parabuthus transvaalicus acts as an alternative type of
venom with distinct mechanism of action, Proc. Natl. Acad. Sci. U. S. A. 100
(2003) 922–927.
[18] Z. Nisani, D.S. Boskovic, S.G. Dunbar, W. Kelln, W.K. Hayes, Investigating the chem-
ical profile of regenerated scorpion (Parabuthus transvaalicus) venom in relation to
metabolic cost and toxicity, Toxicon 60 (2012) 315–323.
[19] A. Bazaa, N. Marrakchi, M. El Ayeb, L. Sanz, J.J. Calvete, Snake venomics: com-
parative analysis of the venom proteomes of the Tunisian snakes Cerastes
cerastes, Cerastes vipera and Macrovipera lebetina, Proteomics 5 (2005)
4223–4235.
[20] P. Juarez, L. Sanz, J.J. Calvete, Snake venomics: characterization of protein families
in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tan-
dem mass spectrometry analysis, Proteomics 4 (2004) 327–338.
[21] A. Menez, R. Stocklin, D. Mebs, ‘Venomics’ or: the venomous systems genome pro-
ject, Toxicon: Off. J. Int. Soc. Toxinol. 47 (2006) 255–259.
[22] D. Biass, A. Violette, N. Hulo, F. Lisacek, P. Favreau, R. Stocklin, Uncovering intense
protein diversification in a cone snail venom gland using an integrative venomics
approach, J. Proteome Res. 14 (2015) 628–638.
[23] V.L. Viala, D. Hildebrand, M. Trusch, T.M. Fucase, J.M. Sciani, D.C. Pimenta, et al.,
Venomics of the Australian eastern brown snake (Pseudonaja textilis): detection
of new venom proteins and splicing variants, Toxicon (2015).
[24] A.M. Maxam, W. Gilbert, A new method for sequencing DNA, Proc. Natl. Acad. Sci.
U. S. A. 74 (1977) 560–564.
[25] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencing with chain-terminating inhib-
itors, Proc. Natl. Acad. Sci. 74 (1977) 5463–5467.
[26] International Human Genome Sequencing C, Finishing the euchromatic sequence
of the human genome, Nature 431 (2004) 931–945.
[27] E.P. Consortium, The ENCODE (ENCyclopedia Of DNA Elements) project, Science
306 (2004) 636–640.
[28] Insights into social insects from the genome of the honeybee Apis mellifera, Nature
443 (2006) 931–949.
[29] J. Gauldie, J. Hanson, F. Rumjanek, R. Shipolini, C. Vernon, The peptide components
of bee venom, Eur. J. Biochem. 61 (1976) 369–376.
[30] N.H. Putnam, M. Srivastava, U. Hellsten, B. Dirks, J. Chapman, A. Salamov, et al., Sea
anemone genome reveals ancestral eumetazoan gene repertoire and genomic or-
ganization, Science 317 (2007) 86–94.
[31] Y. Moran, M. Gurevitz, When positive selection of neurotoxin genes is missing.
The riddle of the sea anemone Nematostella vectensis, FEBS J. 273 (2006)
3886–3892.
69
[32] Y. Moran, D. Praher, A. Schlesinger, A. Ayalon, Y. Tal, U. Technau, Analysis of soluble
protein contents from the nematocysts of a model sea anemone sheds light on
venom evolution, Mar. Biotechnol. (NY) 15 (2013) 329–339.
[33] Y. Moran, H. Weinberger, J.C. Sullivan, A.M. Reitzel, J.R. Finnerty, M. Gurevitz, Con-
certed evolution of sea anemone neurotoxin genes is revealed through analysis of
the Nematostella vectensis genome, Mol. Biol. Evol. 25 (2008) 737–747.
[34] J.A. Chapman, E.F. Kirkness, O. Simakov, S.E. Hampson, T. Mitros, T. Weinmaier,
et al., The dynamic genome of hydra, Nature 464 (2010) 592–596.
[35] C. Shinzato, E. Shoguchi, T. Kawashima, M. Hamada, K. Hisata, M. Tanaka, et al.,
Using the Acropora digitifera genome to understand coral responses to environ-
mental change, Nature 476 (2011) 320–323.
[36] W.C. Warren, L.W. Hillier, J.A. Marshall Graves, E. Birney, C.P. Ponting, F. Grutzner,
et al., Genome analysis of the platypus reveals unique signatures of evolution, Na-
ture 453 (2008) 175–183.
[37] J.H. Werren, S. Richards, C.A. Desjardins, O. Niehuis, J. Gadau, J.K. Colbourne, et al.,
Functional and evolutionary insights from the genomes of three parasitoid Nasonia
species, Science 327 (2010) 343–348.
[38] E.O. Martinson, D. Wheeler, J. Wright, Mrinalini, A.L. Siebert, J.H. Werren, Nasonia
vitripennis venom causes targeted gene expression changes in its fly host, Mol.
Ecol. 23 (2014) 5918–5930.
[39] D.C. de Graaf, M. Aerts, M. Brunain, C.A. Desjardins, F.J. Jacobs, J.H. Werren, et al.,
Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis
from bioinformatic and proteomic studies, Insect Mol. Biol. 19 (Suppl. 1) (2010)
11–26.
[40] M. Kircher, J. Kelso, High-throughput DNA sequencing—concepts and limitations,
BioEssays 32 (2010) 524–536.
[41] Z. Cao, Y. Yu, Y. Wu, P. Hao, Z. Di, Y. He, et al., The genome of Mesobuthus
martensii reveals a unique adaptation model of arthropods, Nat. Commun. 4
(2013) 2602.
[42] F.J. Vonk, N.R. Casewell, C.V. Henkel, A.M. Heimberg, H.J. Jansen, R.J. McCleary, et al.,
The king cobra genome reveals dynamic gene evolution and adaptation in the
snake venom system, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 20651–20656.
[43] K.W. Sanggaard, J.S. Bechsgaard, X. Fang, J. Duan, T.F. Dyrlund, V. Gupta, et al., Spi-
der genomes provide insight into composition and evolution of venom and silk,
Nat. Commun. 5 (2014).
[44] R. Bonasio, G. Zhang, C. Ye, N.S. Mutti, X. Fang, N. Qin, et al., Genomic comparison of
the ants Camponotus floridanus and Harpegnathos saltator, Science 329 (2010)
1068–1071.
[45] K.M. Kapheim, H. Pan, C. Li, S.L. Salzberg, D. Puiu, T. Magoc, et al., Social evolution.
Genomic signatures of evolutionary transitions from solitary to group living, Sci-
ence 348 (2015) 1139–1143.
[46] D.R. Hoffman, Ant venoms, Curr. Opin. Allergy Clin. Immunol. 10 (2010) 342–346.
[47] Y. Wurm, J. Wang, O. Riba-Grognuz, M. Corona, S. Nygaard, B.G. Hunt, et al., The ge-
nome of the fire ant Solenopsis invicta, Proc. Natl. Acad. Sci. U. S. A. 108 (2011)
5679–5684.
[48] S. Kozlov, A. Malyavka, B. McCutchen, A. Lu, E. Schepers, R. Herrmann, et al., A novel
strategy for the identification of toxin-like structures in spider venom, Proteins 59
(2005) 131–140.
[49] B.G. Mille, S. Peigneur, R. Predel, J. Tytgat, Transcriptomic approach reveals the mo-
lecular diversity of Hottentotta conspersus (Buthidae) venom, Toxicon 99 (2015)
73–79.
[50] D. Morgenstern, B.H. Rohde, G.F. King, T. Tal, D. Sher, E. Zlotkin, The tale of a resting
gland: transcriptome of a replete venom gland from the scorpion Hottentotta
judaicus, Toxicon 57 (2011) 695–703.
[51] E.F. Schwartz, R.C. Rodriguez de La Vega, L.D. Possani, E. Diego-Garcia, Tran-
scriptome analysis of the venom gland of the Mexican scorpion Hadrurus gertschi
(Arachnida: Scorpiones), BMC Genomics 8 (2007) 119.
[52] Z. Wang, M. Gerstein, M. Snyder, RNA-Seq: a revolutionary tool for transcriptomics,
Nat. Rev. Genet. 10 (2009) 57–63.
[53] I.M. Francischetti, J.G. Valenzuela, J.F. Andersen, T.N. Mather, J.M. Ribeiro,
Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the
salivary gland of the tick, Ixodes scapularis: identification of factor X and factor
Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex, Blood 99
(2002) 3602–3612.
[54] J. Macrander, M.R. Brugler, M. Daly, A RNA-seq approach to identify putative toxins
from acrorhagi in aggressive and non-aggressive Anthopleura elegantissima polyps,
BMC Genomics 16 (2015) 221.
[55] B.M. von Reumont, A. Blanke, S. Richter, F. Alvarez, C. Bleidorn, R.A. Jenner, The first
venomous crustacean revealed by transcriptomics and functional morphology:
remipede venom glands express a unique toxin cocktail dominated by enzymes
and a neurotoxin, Mol. Biol. Evol. 31 (2014) 48–58.
[56] B.M. von Reumont, L.I. Campbell, S. Richter, L. Hering, D. Sykes, J. Hetmank,
et al., A polychaete's powerful punch: venom gland transcriptomics of Glycera
reveals a complex cocktail of toxin homologs, Genome Biol. Evol. 6 (2014)
2406–2423.
[57] C.M. Whittington, A.T. Papenfuss, D.P. Locke, E.R. Mardis, R.K. Wilson, S. Abubucker,
et al., Novel venom gene discovery in the platypus, Genome Biol. 11 (2010) R95.
[58] B. von Reumont, L. Campbell, R. Jenner, Quo vadis venomics? A roadmap to
neglected venomous invertebrates, Toxins 6 (2014) 3488–3551.
[59] J. Durban, A. Perez, L. Sanz, A. Gomez, F. Bonilla, S. Rodriguez, et al., Integrated
“omics” profiling indicates that miRNAs are modulators of the ontogenetic
venom composition shift in the central American rattlesnake, Crotalus simus
simus, BMC Genomics 14 (2013) 234.
[60] J. Durban, P. Juarez, Y. Angulo, B. Lomonte, M. Flores-Diaz, A. Alape-Giron, et al.,
Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrose-
quencing, BMC Genomics 12 (2011) 259.
70 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
[61] H.L. Gibbs, L. Sanz, M.G. Sovic, J.J. Calvete, Phylogeny-based comparative analysis of
venom proteome variation in a clade of rattlesnakes (Sistrurus sp.), PLoS ONE 8
(2013), e67220.
[62] D. Baek, J. Villen, C. Shin, F.D. Camargo, S.P. Gygi, D.P. Bartel, The impact of
microRNAs on protein output, Nature 455 (2008) 64–71.
[63] M. Selbach, B. Schwanhausser, N. Thierfelder, Z. Fang, R. Khanin, N. Rajewsky,
Widespread changes in protein synthesis induced by microRNAs, Nature 455
(2008) 58–63.
[64] H.L. Gibbs, L. Sanz, J.E. Chiucchi, T.M. Farrell, J.J. Calvete, Proteomic analysis of onto-
genetic and diet-related changes in venom composition of juvenile and adult
dusky pigmy rattlesnakes (Sistrurus miliarius barbouri), J. Proteome 74 (2011)
2169–2179.
[65] P. Escoubas, G. Corzo, B.J. Whiteley, M.-L. Célérier, T. Nakajima, Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry and high-performance
liquid chromatography study of quantitative and qualitative variation in tarantula
spider venoms, Rapid Commun. Mass Spectrom. 16 (2002) 403–413.
[66] D. Chang, A.M. Olenzek, T.F. Duda Jr., Effects of geographical heterogeneity in
species interactions on the evolution of venom genes, Proc. Biol. Sci. 282 (2015).
[67] S. Richier, M. Rodriguez-Lanetty, C.E. Schnitzler, V.M. Weis, Response of the
symbiotic cnidarian Anthopleura elegantissima transcriptome to temperature
and UV increase, Comp. Biochem. Physiol. Part D Genomics Proteomics 3
(2008) 283–289.
[68] D.J. Orts, S. Peigneur, B. Madio, J.S. Cassoli, G.G. Montandon, A.M. Pimenta, et al.,
Biochemical and electrophysiological characterization of two sea anemone type 1
potassium toxins from a geographically distant population of Bunodosoma
caissarum, Mar. Drugs 11 (2013) 655–679.
[69] K. Sunagar, E.A. Undheim, H. Scheib, E.C. Gren, C. Cochran, C.E. Person, et al., Intra-
specific venom variation in the medically significant southern Pacific rattlesnake
(Crotalus oreganus helleri): biodiscovery, clinical and evolutionary implications, J.
Proteome 99 (2014) 68–83.
[70] S. Peigneur, L. Beress, C. Moller, F. Mari, W.G. Forssmann, J. Tytgat, A natural point
mutation changes both target selectivity and mechanism of action of sea anemone
toxins, FASEB J.: Off. Publ. Fed. Am. Soc. Exp. Biol. 26 (2012) 5141–5151.
[71] M.G. Grabherr, B.J. Haas, M. Yassour, J.Z. Levin, D.A. Thompson, I. Amit, et al., Full-
length transcriptome assembly from RNA-Seq data without a reference genome,
Nat. Biotechnol. 29 (2011) 644–652.
[72] J. Archer, G. Whiteley, N.R. Casewell, R.A. Harrison, S.C. Wagstaff, VTBuilder: a tool
for the assembly of multi isoform transcriptomes, BMC Bioinf. 15 (2014) 389.
[73] C.G. Elsik, K.C. Worley, A.K. Bennett, M. Beye, F. Camara, C.P. Childers, et al., Finding
the missing honey bee genes: lessons learned from a genome upgrade, BMC
Genomics 15 (2014) 86.
[74] T. Laver, J. Harrison, P.A. O'Neill, K. Moore, A. Farbos, K. Paszkiewicz, et al., Assessing
the performance of the Oxford nanopore technologies MinION, Biomol. Detect.
Quantif. 3 (2015) 1–8.
[75] A.C. English, S. Richards, Y. Han, M. Wang, V. Vee, J. Qu, et al., Mind the gap:
upgrading genomes with Pacific biosciences RS long-read sequencing technology,
PLoS ONE 7 (2012), e47768.
[76] M.A. Madoui, S. Engelen, C. Cruaud, C. Belser, L. Bertrand, A. Alberti, et al., Genome
assembly using nanopore-guided long and error-free DNA reads, BMC Genomics
16 (2015) 327.
[77] H. Tilgner, F. Grubert, D. Sharon, M.P. Snyder, Defining a personal, allele-specific,
and single-molecule long-read transcriptome, Proc. Natl. Acad. Sci. U. S. A. 111
(2014) 9869–9874.
[78] C.B. Burge, S. Karlin, Finding the genes in genomic DNA, Curr. Opin. Struct. Biol. 8
(1998) 346–354.
[79] M. Yandell, D. Ence, A beginner's guide to eukaryotic genome annotation, Nat. Rev.
Genet. 13 (2012) 329–342.
[80] J.M. Cherry, E.L. Hong, C. Amundsen, R. Balakrishnan, G. Binkley, E.T. Chan, et al.,
Saccharomycesgenome database: the genomics resource of budding yeast, Nucleic
Acids Res. 40 (2012) D700–D705.
[81] G. dos Santos, A.J. Schroeder, J.L. Goodman, V.B. Strelets, M.A. Crosby, J. Thurmond,
et al., FlyBase: introduction of the Drosophila melanogasterrelease 6 reference ge-
nome assembly and large-scale migration of genome annotations, Nucleic Acids
Res. 43 (2015) D690–D697.
[82] T.W. Harris, J. Baran, T. Bieri, A. Cabunoc, J. Chan, W.J. Chen, et al., WormBase 2014:
new views of curated biology, Nucleic Acids Res. 42 (2014) D789–D793.
[83] V. Herzig, D.L. Wood, F. Newell, P.A. Chaumeil, Q. Kaas, G.J. Binford, et al.,
ArachnoServer 2.0, an updated online resource for spider toxin sequences and
structures, Nucleic Acids Res. 39 (2011) D653–D657.
[84] F. Jungo, L. Bougueleret, I. Xenarios, S. Poux, The UniProtKB/Swiss-Prot Tox-Prot
program: a central hub of integrated venom protein data, Toxicon 60 (2012)
551–557.
[85] Y. Terrat, F. Ducancel, Are there unequivocal criteria to label a given protein as a
toxin? Permissive versus conservative annotation processes, Genome Biol. 14
(2013) 406.
[86] A.D. Hargreaves, M.T. Swain, D.W. Logan, J.F. Mulley, Testing the toxicofera: com-
parative transcriptomics casts doubt on the single, early evolution of the reptile
venom system, Toxicon 92 (2014) 140–156.
[87] B.G. Fry, N. Vidal, J.A. Norman, F.J. Vonk, H. Scheib, S.F. Ramjan, et al., Early evolution
of the venom system in lizards and snakes, Nature 439 (2006) 584–588.
[88] N. Vidal, S.B. Hedges, Higher-level relationships of caenophidian snakes inferred
from four nuclear and mitochondrial genes, C. R. Biol. 325 (2002) 987–995.
[89] L. Cohen, N. Lipstein, D. Gordon, Allosteric interactions between scorpion toxin re-
ceptor sites on voltage-gated Na channels imply a novel role for weakly active
components in arthropod venom, FASEB J.: Off. Publ. Fed. Am. Soc. Exp. Biol. 20
(2006) 1933–1935.
[90] S. Milde, G. Hemmrich, F. Anton-Erxleben, K. Khalturin, J. Wittlieb, T.C. Bosch,
Characterization of taxonomically restricted genes in a phylum-restricted cell
type, Genome Biol. 10 (2009) R8.
[91] N.R. Casewell, S.C. Wagstaff, W. Wüster, D.A.N. Cook, F.M.S. Bolton, S.I. King, et al.,
Medically important differences in snake venom composition are dictated by
distinct postgenomic mechanisms, Proc. Natl. Acad. Sci. U. S. A. 111 (2014)
9205–9210.
[92] T. Maier, A. Schmidt, M. Güell, S. Kühner, A.-C. Gavin, R. Aebersold, et al., Quantifi-
cation of mRNA and protein and integration with protein turnover in a bacterium,
Mol. Syst. Biol. 7 (2011) 511.
[93] R.C. Taylor, B.-J.M. Webb Robertson, L.M. Markillie, M.H. Serres, B.E. Linggi, J.T.
Aldrich, et al., Changes in translational efficiency is a dominant regulatory mecha-
nism in the environmental response of bacteria, Integr. Biol. (Camb) 5 (2013)
1393–1406.
[94] Z.L. Bergeron, J.B. Chun, M.R. Baker, D.W. Sandall, S. Peigneur, P.Y.C. Yu, et al., A
‘conovenomic’ analysis of the milked venom from the mollusk-hunting cone
snail conus textile—the pharmacological importance of post-translational modifi-
cations, Peptides 49 (2013) 145–158.
[95] M. Rong, Z. Duan, J. Chen, J. Li, Y. Xiao, S. Liang, Native pyroglutamation of
huwentoxin-IV: a post-translational modification that increases the trapping
ability to the sodium channel, PLoS ONE 8 (2013), e65984.
[96] E. Kapp, F. Schütz, Overview of tandem mass spectrometry (MS/MS) database
search algorithms, Curr. Protoc. Protein Sci. (2007) Chapter 25:Unit25.2.
[97] A.I. Nesvizhskii, Protein identification by tandem mass spectrometry and sequence
database searching, Methods Mol. Biol. 367 (2007) 87–119.
[98] A.T. Tu, B.L. Adams, Phylogenetic relationships among venomous snakes of the
genus Agkistrodon from Asia and the North American continent, Nature 217
(1968) 760–762.
[99] C.C. Yang, Chemistry and evolution of toxins in snake venoms, Toxicon: Off. J. Int.
Soc. Toxinol. 12 (1974) 1–43.
[100] E.M. Bettke, D.D. Watt, T. Tu, Electrophoretic patterns of venoms from species
of Crotalidae and Elapidae snakes, Toxicon: Off. J. Int. Soc. Toxinol. 4 (1966)
73–76.
[101] C.A. Bonilla, N.V. Horner, Comparative electrophoresis of Crotalus and Agkistrodon
venoms from North American snakes, Toxicon: Off. J. Int. Soc. Toxinol. 7 (1969)
327–329.
[102] R. Foote, J.A. MacMahon, Electrophoretic studies of rattlesnake (Crotalus &
Sistrurus) venom: taxonomic implications, Comp. Biochem. Physiol. B 57 (1977)
235–241.
[103] A. Yahel-Niv, E. Zlotkin, Comparative studies on venom obtained from individ-
ual scorpions by natural stings, Toxicon: Off. J. Int. Soc. Toxinol. 17 (1979)
435–446.
[104] N.J. da Silva Júnior, P.R. Griffin, S.D. Aird, Comparative chromatography of
Brazilian coral snake (Micrurus) venoms, Comp. Biochem. Physiol. B 100
(1991) 117–126.
[105] J.L. Glenn, R.C. Straight, Intergradation of two different venom populations of the
Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona, Toxicon: Off. J. Int.
Soc. Toxinol. 27 (1989) 411–418.
[106] M.F. Martin, H. Rochat, P. Marchot, P.E. Bougis, Use of high performance liquid
chromatography to demonstrate quantitative variation in components of venom
from the scorpion Androctonus australisHector, Toxicon: Off. J. Int. Soc. Toxinol.
25 (1987) 569–573.
[107] J.G. Soto, J.C. Perez, M.M. Lopez, M. Martinez, T.B. Quintanilla-Hernandez, M.S.
Santa-Hernandez, et al., Comparative enzymatic study of HPLC-fractionated
Crotalus venoms, Comp. Biochem. Physiol. B 93 (1989) 847–855.
[108] L.F. Machado, S. Laugesen, E.D. Botelho, C.A.O. Ricart, W. Fontes, K.C. Barbaro, et al.,
Proteome analysis of brown spider venom: identification of loxnecrogin isoforms
in Loxosceles gaucho venom, Proteomics 5 (2005) 2167–2176.
[109] M.C. Menezes, M.F. Furtado, S.R. Travaglia-Cardoso, A.C.M. Camargo, S.M.T. Serrano,
Sex-based individual variation of snake venom proteome among eighteen Bothrops
jararaca siblings, Toxicon: Off. J. Int. Soc. Toxinol. 47 (2006) 304–312.
[110] S.M.T. Serrano, J.D. Shannon, D. Wang, A.C.M. Camargo, J.W. Fox, A multiface-
ted analysis of viperid snake venoms by two-dimensional gel electrophoresis:
an approach to understanding venom proteomics, Proteomics 5 (2005)
501–510.
[111] J. Vejayan, T.L. Khoon, H. Ibrahim, Comparative analysis of the venom proteome of
four important Malaysian snake species, J. Venom. Anim. Toxins. Incl. Trop. Dis. 20
(2014) 6.
[112] J.C. Daltry, G. Ponnudurai, C.K. Shin, N.H. Tan, R.S. Thorpe, W. Wüster, Electropho-
retic profiles and biological activities: intraspecific variation in the venom of the
Malayan pit viper (Calloselasma rhodostoma), Toxicon: Off. J. Int. Soc. Toxinol. 34
(1996) 67–79.
[113] H.L. Gibbs, L. Sanz, J.J. Calvete, Snake population venomics: proteomics-based anal-
yses of individual variation reveals significant gene regulation effects on venom
protein expression in Sistrurus rattlesnakes, J. Mol. Evol. 68 (2009) 113–125.
[114] P. Kleinert, T. Kuster, D. Arnold, J. Jaeken, C.W. Heizmann, H. Troxler, Effect of
glycosylation on the protein pattern in 2-D-gel electrophoresis, Proteomics 7
(2007) 15–22.
[115] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, et al., Protein and poly-
mer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrom-
etry, Rapid Commun. Mass Spectrom. 2 (1988) 151–153.
[116] L.M. Smith, N.L. Kelleher, Consortium for top down P. Proteoform: a single term de-
scribing protein complexity, Nat. Methods 10 (2013) 186–187.
[117] J.R. Perkins, C.E. Parker, K.B. Tomer, The characterization of snake venoms using
capillary electrophoresis in conjunction with electrospray mass spectrometry:
black mambas, Electrophoresis 14 (1993) 458–468.
 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72
[118] J.R. Perkins, B. Smith, R.T. Gallagher, D.S. Jones, S.C. Davis, A.D. Hoffman, et al.,
Application of electrospray mass spectrometry and matrix-assisted laser de-
sorption ionization time-of-flight mass spectrometry for molecular weight as-
signment of peptides in complex mixtures, J. Am. Soc. Mass Spectrom. 4 (1993)
670–684.
[119] P. Favreau, L. Menin, S. Michalet, F. Perret, O. Cheneval, M. Stöcklin, et al., Mass
spectrometry strategies for venom mapping and peptide sequencing from crude
venoms: case applications with single arthropod specimen, Toxicon: Off. J. Int.
Soc. Toxinol. 47 (2006) 676–687.
[120] D.G. Nascimento, B. Rates, D.M. Santos, T. Verano-Braga, A. Barbosa-Silva, A.A.A.
Dutra, et al., Moving pieces in a taxonomic puzzle: venom 2D-LC/MS and data
clustering analyses to infer phylogenetic relationships in some scorpions
from the Buthidae family (Scorpiones), Toxicon: Off. J. Int. Soc. Toxinol. 47
(2006) 628–639.
[121] H. Steen, M. Mann, The ABC's (and XYZ's) of peptide sequencing, Nat. Rev. Mol. Cell
Biol. 5 (2004) 699–711.
[122] A. Alape-Girón, L. Sanz, J. Escolano, M. Flores-Díaz, M. Madrigal, M. Sasa, et al.,
Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual,
and ontogenetic variations, J. Proteome Res. 7 (2008) 3556–3571.
[123] A.M. Rodriguez, S. Dutertre, R.J. Lewis, F. Marí, Intraspecific variations in Conus
purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-triple TOF-MS,
Anal. Bioanal. Chem. (2015).
[124] J.J. Calvete, P. Ghezellou, O. Paiva, T. Matainaho, A. Ghassempour, H. Goudarzi, et al.,
Snake venomics of two poorly known Hydrophiinae: comparative proteomics of
the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis
cyanocinctus, J. Proteome 75 (2012) 4091–4101.
[125] B. Nguyen, J. Molgó, H. Lamthanh, E. Benoit, T.A. Khuc, D.N. Ngo, et al., High accu-
racy mass spectrometry comparison of Conus bandanus and Conus marmoreus
venoms from the South Central coast of Vietnam, Toxicon: Off. J. Int. Soc. Toxinol.
75 (2013) 148–159.
[126] A.K. Tashima, L. Sanz, A.C.M. Camargo, S.M.T. Serrano, J.J. Calvete, Snake venomics
of the Brazilian pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of
taxonomy markers, J. Proteome 71 (2008) 473–485.
[127] E.A.B. Undheim, K. Sunagar, B.R. Hamilton, A. Jones, D.J. Venter, B.G. Fry, et al., Mul-
tifunctional warheads: diversification of the toxin arsenal of centipedes via novel
multidomain transcripts, J. Proteome 102 (2014) 1–10.
[128] A. Zelanis, A.K. Tashima, A.F.M. Pinto, A.F. Paes Leme, D.R. Stuginski, M.F. Furtado,
et al., Bothrops jararaca venom proteome rearrangement upon neonate to adult
transition, Proteomics 11 (2011) 4218–4228.
[129] D.C. Pimenta, B.C. Prezoto, K. Konno, R.L. Melo, M.F. Furtado, A.C.M. Camargo,
et al., Mass spectrometric analysis of the individual variability of Bothrops
jararaca venom peptide fraction. Evidence for sex-based variation among the
bradykinin-potentiating peptides, Rapid Commun. Mass Spectrom. 21 (2007)
1034–1042.
[130] C.T. Cologna, J.S. Cardoso, E. Jourdan, M. Degueldre, G. Upert, N. Gilles, et al.,
Peptidomic comparison and characterization of the major components of the
venom of the giant ant Dinoponera quadriceps collected in four different areas of
Brazil, J. Proteome 94 (2013) 413–422.
[131] K.Y. Tan, C.H. Tan, S.Y. Fung, N.H. Tan, Venomics, lethality and neutralization of
Naja kaouthia (monocled cobra) venoms from three different geographical regions
of Southeast Asia, J. Proteome 120 (2015) 105–125.
[132] L. Sanz, H.L. Gibbs, S.P. Mackessy, J.J. Calvete, Venom proteomes of closely relat-
ed Sistrurus rattlesnakes with divergent diets, J. Proteome Res. 5 (2006)
2098–2112.
[133] G.D. Findlay, M.J. MacCoss, W.J. Swanson, Proteomic discovery of previously unan-
notated, rapidly evolving seminal fluid genes in Drosophila, Genome Res. 19 (2009)
886–896.
[134] S.D. Robinson, H. Safavi-Hemami, S. Raghuraman, J.S. Imperial, A.T.
Papenfuss, R.W. Teichert, et al., Discovery by proteogenomics and character-
ization of an RF-amide neuropeptide from cone snail venom, J. Proteome 114
(2015) 38–47.
[135] K. Krug, S. Popic, A. Carpy, C. Taumer, B. Macek, Construction and assessment of in-
dividualized proteogenomic databases for large-scale analysis of nonsynonymous
single nucleotide variants, Proteomics 14 (2014) 2699–2708.
[136] A.D. Catherman, O.S. Skinner, N.L. Kelleher, Top down proteomics: facts and per-
spectives, Biochem. Biophys. Res. Commun. 445 (2014) 683–693.
[137] I. Karbat, M. Turkov, L. Cohen, R. Kahn, D. Gordon, M. Gurevitz, et al., X-ray struc-
ture and mutagenesis of the scorpion depressant toxin LqhIT2 reveals key determi-
nants crucial for activity and anti-insect selectivity, J. Mol. Biol. 366 (2007)
586–601.
[138] P. Anand, A. Grigoryan, M.H. Bhuiyan, B. Ueberheide, V. Russell, J. Quinoñez, et al.,
Sample limited characterization of a novel disulfide-rich venom peptide toxin from
terebrid marine snail Terebra variegata, PLoS ONE 9 (2014), e94122.
[139] B.M. Ueberheide, D. Fenyö, P.F. Alewood, B.T. Chait, Rapid sensitive analysis of cys-
teine rich peptide venom components, Proc. Natl. Acad. Sci. U. S. A. 106 (2009)
6910–6915.
[140] D. Petras, P. Heiss, R.D. Süssmuth, J.J. Calvete, Venom proteomics of Indonesian king
cobra, Ophiophagus hannah: integrating top-down and bottom-up approaches, J.
Proteome Res. 14 (2015) 2539–2556.
[141] M. Bern, Y. Cai, D. Goldberg, Lookup peaks: a hybrid of de novo sequencing and da-
tabase search for protein identification by tandem mass spectrometry, Anal. Chem.
79 (2007) 1393–1400.
[142] L. Zamdborg, R.D. LeDuc, K.J. Glowacz, Y.-B. Kim, V. Viswanathan, I.T.
Spaulding, et al., ProSight PTM 2.0: improved protein identification and char-
acterization for top down mass spectrometry, Nucleic Acids Res. 35 (2007)
W701–W706.
71
[143] H. Chi, H. Chen, K. He, L. Wu, B. Yang, R.-X. Sun, et al., pNovo+: de novo peptide
sequencing using complementary HCD and ETD tandem mass spectra, J. Proteome
Res. 12 (2013) 615–625.
[144] K. Jeong, S. Kim, P.A. Pevzner, UniNovo: a universal tool for de novo peptide
sequencing, Bioinformatics (Oxford, England) 29 (2013) 1953–1962.
[145] Y. Yan, A. Kusalik, F.-X. Wu, A framework of de novo peptide sequencing for
multiple tandem mass spectra, IEEE Trans. Nanobioscience. (2015).
[146] B. Ma, K. Zhang, C. Hendrie, C. Liang, M. Li, A. Doherty-Kirby, et al., PEAKS: powerful
software for peptide de novo sequencing by tandem mass spectrometry, Rapid
Commun. Mass Spectrom.: RCM 17 (2003) 2337–2342.
[147] A. Guthals, K.R. Clauser, N. Bandeira, Shotgun protein sequencing with meta-contig
assembly, Mol. Cell. Proteomics 11 (2012) 1084–1096.
[148] X. Liu, L.J.M. Dekker, S. Wu, M.M. Vanduijn, T.M. Luider, N. Tolić, et al., De novo pro-
tein sequencing by combining top-down and bottom-up tandem mass spectra, J.
Proteome Res. 13 (2014) 3241–3248.
[149] R. Datta, M. Bern, Spectrum fusion: using multiple mass spectra for de novo pep-
tide sequencing, J. Comput. Biol. 16 (2009) 1169–1182.
[150] J. Cascardi, B.A. Young, H.D. Husic, J. Sherma, Protein variation in the venom spat by
the red spitting cobra, Naja pallida (Reptilia: Serpentes), Toxicon: Off. J. Int. Soc.
Toxinol. 37 (1999) 1271–1279.
[151] C.A. Prator, K.M. Murayama, J.R. Schulz, Venom variation during prey capture by
the cone snail, Conus textile, PLoS ONE 9 (2014), e98991.
[152] E.B. Undheim, D.N. Georgieva, H.H. Thoen, J.A. Norman, J. Mork, C. Betzel, et al.,
Venom on ice: first insights into Antarctic octopus venoms, Toxicon: Off. J. Int.
Soc. Toxinol. 56 (2010) 897–913.
[153] B. Chatterji, A. Pich, MALDI imaging mass spectrometry and analysis of endogenous
peptides, Expert Rev. Proteomics 10 (2013) 381–388.
[154] M.M. Gessel, J.L. Norris, R.M. Caprioli, MALDI imaging mass spectrometry: spatial
molecular analysis to enable a new age of discovery, J. Proteome 107 (2014)
71–82.
[155] J. Stauber, L. MacAleese, J. Franck, E. Claude, M. Snel, B.K. Kaletas, et al., On-tissue
protein identification and imaging by MALDI-ion mobility mass spectrometry, J.
Am. Soc. Mass Spectrom. 21 (2010) 338–347.
[156] A. Kiss, D.F. Smith, B.R. Reschke, M.J. Powell, R.M.A. Heeren, Top-down mass spec-
trometry imaging of intact proteins by laser ablation ESI FT-ICR MS, Proteomics 14
(2014) 1283–1289.
[157] E.A.B. Undheim, B.R. Hamilton, N.D. Kurniawan, G. Bowlay, B.W. Cribb, D.J. Merritt,
et al., Production and packaging of a biological arsenal: evolution of centipede
venoms under morphological constraint, Proc. Natl. Acad. Sci. U. S. A. 112 (2015)
4026–4031.
[158] G. Brunda, B.S. Rao, R.K. Sarin, Quantitation of Indian krait (Bungarus caeruleus)
venom in human specimens of forensic origin by indirect competitive inhibition
enzyme-linked immunosorbent assay, J. AOAC Int. 89 (2006) 1360–1366.
[159] Z.E. Selvanayagam, S.G. Gnanavendhan, K.A. Ganesh, D. Rajagopal, P.V. Rao, ELISA
for the detection of venoms from four medically important snakes of India,
Toxicon: Off. J. Int. Soc. Toxinol. 37 (1999) 757–770.
[160] N.H. Tan, K.H. Yeo, M.I. Jaafar, The use of enzyme-linked immunosorbent
assay for the quantitation of Calloselasma rhodostoma (Malayan pit viper)
venom and venom antibodies, Toxicon: Off. J. Int. Soc. Toxinol. 30 (1992)
1609–1620.
[161] S.D. Aird, N.J. da Silva, Comparative enzymatic composition of Brazilian coral snake
(Micrurus) venoms, Comp. Biochem. Physiol. B 99 (1991) 287–294.
[162] L.M. Mulfinger, A.W. Benton, M.W. Guralnick, R.A. Wilson, A qualitative and quan-
titative analysis of proteins found in vespid venoms, J. Allergy Clin. Immunol. 77
(1986) 681–686.
[163] S. Namiranian, R.C. Hider, Use of HPLC to demonstrate variation of venom toxin
composition in the Thailand cobra venoms Naja naja kaouthia and Naja naja
siamensis, Toxicon: Off. J. Int. Soc. Toxinol. 30 (1992) 47–61.
[164] H. Arikan, N. Alpagut Keskin, I.E. Cevik, C. Ilgaz, Age-dependent variations in the
venom proteins of Vipera xanthina (Gray, 1849) (Ophidia: Viperidae), Türk.
Parazitol. Derg. 30 (2006) 163–165.
[165] M. Lang Balija, A. Vrdoljak, L. Habjanec, B. Dojnović, B. Halassy, B. Vranesić, et al., The
variability of Vipera ammodytes ammodytes venoms from Croatia—biochemical
properties and biological activity, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 140
(2005) 257–263.
[166] M. Bantscheff, S. Lemeer, M.M. Savitski, B. Kuster, Quantitative mass spectrometry
in proteomics: critical review update from 2007 to the present, Anal. Bioanal.
Chem. 404 (2012) 939–965.
[167] S.W. Holman, P.F.G. Sims, C.E. Eyers, The use of selected reaction monitoring in
quantitative proteomics, Bioanalysis 4 (2012) 1763–1786.
[168] J.-F. Gao, Y.-F. Qu, X.-Q. Zhang, Y. He, X. Ji, Neonate-to-adult transition of snake
venomics in the short-tailed pit viper, Gloydius brevicaudus, J. Proteome 84
(2013) 148–157.
[169] S.A. Ali, K. Baumann, T.N.W. Jackson, K. Wood, S. Mason, E.A.B. Undheim, et al.,
Proteomic comparison of Hypnale hypnale (hump-nosed pit-viper) and
Calloselasma rhodostoma (Malayan pit-viper) venoms, J. Proteome 91 (2013)
338–343.
[170] H. Safavi-Hemami, H. Hu, D.G. Gorasia, P.K. Bandyopadhyay, P.D. Veith, N.D. Young,
et al., Combined proteomic and transcriptomic interrogation of the venom gland of
Conus geographus uncovers novel components and functional compartmentaliza-
tion, Mol. Cell. Proteomics 13 (2014) 938–953.
[171] X. Xu, Z. Duan, Z. Di, Y. He, J. Li, Z. Li, et al., Proteomic analysis of the venom from
the scorpion Mesobuthus martensii, J. Proteome 106 (2014) 162–180.
[172] T. Rachamim, D. Morgenstern, D. Aharonovich, V. Brekhman, T. Lotan, D. Sher, The
dynamically evolving nematocyst content of an anthozoan, a scyphozoan, and a
hydrozoan, Mol. Biol. Evol. 32 (2015) 740–753.
72 K. Sunagar et al. / Journal of Proteomics 135 (2016) 62–72

[173] D. Sher, Y. Fishman, M. Zhang, M. Lebendiker, A. Gaathon, J.M. Mancheno, et al., [175] E.S. Wong, D. Morgenstern, E. Mofiz, S. Gombert, K.M. Morris, P. Temple-Smith,
Hydralysins, a new category of beta-pore-forming toxins in Cnidaria, J. Biol. et al., Proteomics and deep sequencing comparison of seasonally active venom
Chem. 280 (2005) 22847–22855. glands in the platypus reveals novel venom peptides and distinct expression pro-
[174] Y. Moran, H. Weinberger, A.M. Reitzel, J.C. Sullivan, R. Kahn, D. Gordon, et al., Intron files, Mol. Cell. Proteomics 11 (2012) 1354–1364.
retention as a posttranscriptional regulatory mechanism of neurotoxin expression
at early life stages of the starlet anemone Nematostella vectensis, J. Mol. Biol. 380
(2008) 437–443.