Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded ß-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

A novel proline-rich M-superfamily conotoxin that can simultaneously affect sodium, potassium and calcium currents

Background Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. Methods Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. Results About 10 μM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 μM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 μM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 μM. Moreover, 10 μM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. Conclusions Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.(AU)

ICTC-RAAC: An improved web predictor for identifying the types of ion channel-targeted conotoxins by using reduced amino acid cluster descriptors.

Conotoxins are small peptide toxins which are rich in disulfide and have the unique diversity of sequences. It is significant to correctly identify the types of ion channel-targeted conotoxins because that they are considered as the optimal pharmacological candidate medicine in drug design owing to their ability specifically binding to ion channels and interfering with neural transmission. Comparing with other feature extracting methods, the reduced amino acid cluster (RAAC) better resolved in simplifying protein complexity and identifying functional conserved regions. Thus, in our study, 673 RAACs generated from 74 types of reduced amino acid alphabet were comprehensively assessed to establish a state-of-the-art predictor for predicting ion channel-targeted conotoxins. The results showed Type 20, Cluster 9 (T = 20, C = 9) in the tripeptide composition (N = 3) achieved the best accuracy, 89.3%, which was based on the algorithm of amino acids reduction of variance maximization. Further, the ANOVA with incremental feature selection (IFS) was used for feature selection to improve prediction performance. Finally, the cross-validation results showed that the best overall accuracy we calculated was 96.4% and 1.8% higher than the best accuracy of previous studies. Based on the predictor we proposed, a user-friendly webserver was established and can be friendly accessed at http://bioinfor.imu.edu.cn/ictcraac.

LC-Trapped Ion Mobility Spectrometry-TOF MS Differentiation of 2- and 3-Disulfide-Bonded Isomers of the µ-Conotoxin PIIIA.

Disulfide bonds within cysteine-rich peptides are important for their stability and biological function. In this respect, the correct disulfide connectivity plays a decisive role. The differentiation of individual disulfide-bonded isomers by traditional high-performance liquid chromatography (HPLC) and mass spectrometry (MS) is limited due to the similarity in physicochemical properties of the isomers sharing the same amino acid sequence. By using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), several 2- and 3-disulfide-bonded isomers of the µ-conotoxin PIIIA were investigated for their distinguishability by collision cross section (CCS) values and their characteristic mobilogram traces. The isomers could be differentiated by TIMS-MS and also identified in mixing experiments. Thus, TIMS-MS provides a highly valuable and enriching addition to standard HPLC and MS analysis of conformational isomers of disulfide-rich peptides and proteins.

Extraction of α-neurotoxins from equine plasma by receptor based affinity purification.

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α-neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled "Conotoxin", was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α-neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one-step affinity purification method was developed for the detection of α-neurotoxins in plasma.

Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS.

The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple individuals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom analysis requires separation through RP-HPLC followed by MALDI-TOF mass spectrometry or direct analysis using LC-ESI-TripleTOF-MS. Using these techniques, venom intraspecific variability and comparison between injected and dissected were assessed.

PPIase is associated with the diversity of conotoxins from cone snail venom glands.

Cone snails are incredibly rich sources of bioactive conopeptides with potential for use in neuroscience research and novel drug development. In order to investigate the synthesis of diversified conopeptides in venom glands, the proteome and peptidome profiles of conus venom were analyzed using HPLC and mass spectrometry. The peptidome profile of the venom components with a molecular weight under 10 kDa showed that the peptides with unique mass values from the venom glands of Conus caracteristicus, Conus lividus and Conus textile are 188, 413 and 265, respectively, and there are 39 overlapping peptides among the three species. Proteome profiling of the components with molecular weights above 10 kDa showed that the most abundant proteins (38.6%) are involved in metabolism and that approximately 6.8% of proteins are involved in protein synthesis, folding and post-translational modification. Among these proteins, PPIase is one protein identified from C. textile based on proteomic analysis. Conus PPIase was successfully expressed as a fusion protein with TRX in an Escherichia coli expression system for further function study. In-vitro enzyme activity assays showed that cone snail PPIase could induce the cis-trans isomerization of the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The HPLC mapping analyses of linear lt14a, a conotoxin with 3 prolines, showed that different lt14a isoforms appear after incubation with PPIase. Our results suggest that PPIase may modify conotoxins containing prolines and play an important role in the process of peptide folding and modification in venom glands and contribute to conotoxin diversity.

A tyrosine-containing analog of mu-conotoxin GIIIA as ligand in the receptor binding assay for paralytic shellfish poisons.

Development of novel analytical tools to detect marine biotoxins has been warranted in view of the apparent global pervasiveness of algal-derived shellfish poisoning, and the limitations of existing methods. Here, we describe the initial phase in the development and evaluation of a tyrosine-containing analog of µ-conotoxin (µ-CTX) GIIIA as an alternative to saxitoxin (STX) in a receptor binding assay (RBA) for paralytic shellfish poisons. The peptide analog was synthesized and characterized for structure and bioactivity. The major product of oxidation elicited paralytic symptoms in mice at a minimum dose of 1.31 mg kg(-1) (i.p.). Mass spectrometry analysis of the bioactive peptide gave a molecular mass of 2637.52 Da that was close to the predicted value. Iodination via chloramine-T produced non-, mono- and di-iodinated peptides (respectively, NIP, MIP and DIP). Competition assays against (3)H-STX revealed higher Ki and EC50 (P < 0.0001, ANOVA) indicating reduced affinity for the receptor, and limited displacement of receptor-bound STX. However, subsequent use of MIP may extend the application of RBA to detect small changes in toxin levels owing to its likely enhanced displacement by STX. This may be useful in analyzing samples with toxicities near the regulatory limit, or in establishing baseline values in high risk environments.

Systemic effects induced by intralesional injection of ?-conotoxin MVIIC after spinal cord injury in rats

Calcium channel blockers such as conotoxins have shown a great potential to reduce brain and spinal cord injury. MVIIC neuroprotective effects analyzed in in vitro models of brain and spinal cord ischemia suggest a potential role of this toxin in preventing injury after spinal cord trauma. However, previous clinical studies with MVIIC demonstrated that clinical side effects might limit the usefulness of this drug and there is no research on its systemic effects. Therefore, the present study aimed to investigate the potential toxic effects of MVIIC on organs and to evaluate clinical and blood profiles of rats submitted to spinal cord injury and treated with this marine toxin. Rats were treated with placebo or MVIIC (at doses of 15, 30, 60 or 120 pmol) intralesionally following spinal cord injury. Seven days after the toxin administration, kidney, brain, lung, heart, liver, adrenal, muscles, pancreas, spleen, stomach, and intestine were histopathologically investigated. In addition, blood samples collected from the rats were tested for any hematologic or biochemical changes.

Systemic effects induced by intralesional injection of ?-conotoxin MVIIC after spinal cord injury in rats

Calcium channel blockers such as conotoxins have shown a great potential to reduce brain and spinal cord injury. MVIIC neuroprotective effects analyzed in in vitro models of brain and spinal cord ischemia suggest a potential role of this toxin in preventing injury after spinal cord trauma. However, previous clinical studies with MVIIC demonstrated that clinical side effects might limit the usefulness of this drug and there is no research on its systemic effects. Therefore, the present study aimed to investigate the potential toxic effects of MVIIC on organs and to evaluate clinical and blood profiles of rats submitted to spinal cord injury and treated with this marine toxin. Rats were treated with placebo or MVIIC (at doses of 15, 30, 60 or 120 pmol) intralesionally following spinal cord injury. Seven days after the toxin administration, kidney, brain, lung, heart, liver, adrenal, muscles, pancreas, spleen, stomach, and intestine were histopathologically investigated. In addition, blood samples collected from the rats were tested for any hematologic or biochemical changes.

Systematic interrogation of the Conus marmoreus venom duct transcriptome with ConoSorter reveals 158 novel conotoxins and 13 new gene superfamilies.

BACKGROUND: Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining. RESULTS: Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies. CONCLUSIONS: Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.

Expression and secretion of functional recombinant µO-conotoxin MrVIB-His-tag in Escherichia coli.

µO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus.

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.

Specialisation of the venom gland proteome in predatory cone snails reveals functional diversification of the conotoxin biosynthetic pathway.

Conotoxins, venom peptides from marine cone snails, diversify rapidly as speciation occurs. It has been suggested that each species can synthesize between 1000 and 1900 different toxins with little to no interspecies overlap. Conotoxins exhibit an unprecedented degree of post-translational modifications, the most common one being the formation of disulfide bonds. Despite the great diversity of structurally complex peptides, little is known about the glandular proteins responsible for their biosynthesis and maturation. Here, proteomic interrogations on the Conus venom gland led to the identification of novel glandular proteins of potential importance for toxin synthesis and secretion. A total of 161 and 157 proteins and protein isoforms were identified in the venom glands of Conus novaehollandiae and Conus victoriae, respectively. Interspecies differences in the venom gland proteomes were apparent. A large proportion of the proteins identified function in protein/peptide translation, folding, and protection events. Most intriguingly, however, we demonstrate the presence of a multitude of isoforms of protein disulfide isomerase (PDI), the enzyme catalyzing the formation and isomerization of the native disulfide bond. Investigating whether different PDI isoforms interact with distinct toxin families will greatly advance our knowledge on the generation of cone snail toxins and disulfide-rich peptides in general.

Conotoxin protein classification using free scores of words and support vector machines.

BACKGROUND: Conotoxin has been proven to be effective in drug design and could be used to treat various disorders such as schizophrenia, neuromuscular disorders and chronic pain. With the rapidly growing interest in conotoxin, accurate conotoxin superfamily classification tools are desirable to systematize the increasing number of newly discovered sequences and structures. However, despite the significance and extensive experimental investigations on conotoxin, those tools have not been intensively explored. RESULTS: In this paper, we propose to consider suboptimal alignments of words with restricted length. We developed a scoring system based on local alignment partition functions, called free score. The scoring system plays the key role in the feature extraction step of support vector machine classification. In the classification of conotoxin proteins, our method, SVM-Freescore, features an improved sensitivity and specificity by approximately 5.864% and 3.76%, respectively, over previously reported methods. For the generalization purpose, SVM-Freescore was also applied to classify superfamilies from curated and high quality database such as ConoServer. The average computed sensitivity and specificity for the superfamily classification were found to be 0.9742 and 0.9917, respectively. CONCLUSIONS: The SVM-Freescore method is shown to be a useful sequence-based analysis tool for functional and structural characterization of conotoxin proteins. The datasets and the software are available at http://faculty.uaeu.ac.ae/nzaki/SVM-Freescore.htm.

Intraspecies variability and conopeptide profiling of the injected venom of Conus ermineus.

The venom of cone snails (ssp. Conus), a genus of predatory mollusks, is a vast source of bioactive peptides. Conus venom expression is complex, and venom composition can vary considerably depending upon the method of extraction and the species of cone snail in question. The injected venom from Conus ermineus, the only fish-hunting cone snail species that inhabits the Atlantic Ocean, was characterized using nanoNMR spectroscopy, MALDI-TOF mass spectrometry, RP-HPLC and nanoLC-ESI-MS. These methods allowed us to evaluate the variability of the venom within this species. Single specimens of C. ermineus show unchanged injected venom mass spectra and HPLC profiles over time. However, there was significant variability of the injected venom composition from specimen to specimen, in spite of their common biogeographic origin. Using nanoLC-ESI-MS, we determined that over 800 unique conopeptides are expressed by this reduced set of C. ermineus specimens. This number is considerably larger than previous estimates of the molecular repertoire available to cone snails to immobilize prey. These results support the idea of the existence of a complex regulatory mechanism to express specific venom peptides for injection into prey. These intraspecies differences can be a result of a combination of genetic and environmental factors. The differential expression of venom components represents a neurochemical paradigm that warrants further investigation.

Proteomic analysis provides insights on venom processing in Conus textile.

Conus species of marine snails deliver a potent collection of toxins from the venom duct via a long proboscis attached to a harpoon tooth. Conotoxins are known to possess powerful neurological effects and some have been developed for therapeutic uses. Using mass-spectrometry based proteomics, qualitative and quantitative differences in conotoxin components were found in the proximal, central and distal sections of the Conus textile venom duct suggesting specialization of duct sections for biosynthesis of particular conotoxins. Reversed phase HPLC followed by Orbitrap mass spectrometry and data analysis using SEQUEST and ProLuCID identified 31 conotoxin sequences and 25 post-translational modification (PTM) variants with King-Kong 2 peptide being the most abundant. Several previously unreported variants of known conopeptides were found and this is the first time that HyVal is reported for a disulfide rich Conus peptide. Differential expression along the venom duct, production of PTM variants, alternative proteolytic cleavage sites, and venom processing enroute to the proboscis all appear to contribute to enriching the combinatorial pool of conopeptides and producing the appropriate formulation for a particular hunting situation. The complementary tools of mass spectrometry-based proteomics and molecular biology can greatly accelerate the discovery of Conus peptides and provide insights on envenomation and other biological strategies of cone snails.

TxXIIIA, an atypical homodimeric conotoxin found in the Conus textile venom.

Venoms of predatory Conus snails are composed of several hundreds of peptide toxins. Many of these peptides display a high selectivity for particular membrane receptors such as ionic channels or G-protein coupled receptors. This property makes them very promising tools for the study of receptors and potential new drugs. Conus snails synthesize toxins under various folds, each fold related to particular pharmacological activities. Aiming the discovery of new conotoxins, we looked for toxins with original fold in the Conus textile venom by offline LC-MALDI-TOF/TOF mass spectrometry. Venom fractions were analysed by MALDI-TOF (in 2,5-dihydroxybenzoic acid) before and after the "in-solution" reduction of the disulfide bridges. Comparison of the spectra allows the classification of a large number of conotoxins according to the number of disulfide bridges. We focussed on a component at m/z 2785.7 (non-reduced)/ 1398.4 (reduced), which might represent a novel type of homodimeric toxin. The sequence TSDCCFYHNCCC was determined by De novo sequencing on the reduced species and represent a new fold. This sequence has already been described as the C-terminus part of a conotoxin scaffold IX precursor (expasy: Q9BPH1) but the power of our study resides in the fact that mass spectrometry highlights the right length of the toxin as well as its homodimeric form which could not be determined by the previous cDNA study. TxXIIIA is also the first homodimeric conotoxin with five disulfide bonds and composed of two monomers containing an odd number of cysteins.

Comparative proteomic study of the venom of the piscivorous cone snail Conus consors.

In the context of an exhaustive study of the piscivorous cone snail Conus consors, we performed an in-depth analysis of the intact molecular masses that can be detected in the animal's venom, using MALDI and ESI mass spectrometry. We clearly demonstrated that, for the venom of this species at least, it is essential to use both techniques in order to obtain the broadest data set of molecular masses. Only 20% of the total number of molecules detected were found in both mass lists. The two data sets were also compared in terms of mass range and relative hydrophobicity of the components detected in each. With a view to an extensive analysis of this venom's proteome, we further performed a comparative study by ESI-MS between venom obtained after classical dissection of the venom duct versus venom obtained by milking live animals. Surprisingly, although many fewer components were found in the milked venom than in the dissected venom, approximately 50% of those found had not been seen in the dissected venom. Several questions raised by these observations are discussed. With regards to the current knowledge of the cone snail venom composition, our results emphasize the complementary nature of the mass spectrometry methods and of the two techniques used in venom collection.

Snake venomics and antivenomics of Bothrops colombiensis, a medically important pitviper of the Bothrops atrox-asper complex endemic to Venezuela: Contributing to its taxonomy and snakebite management.

The taxonomic status of the medically important pitviper of the Bothrops atrox-asper complex endemic to Venezuela, which has been classified as Bothrops colombiensis, remains incertae cedis. To help resolving this question, the venom proteome of B. colombiensis was characterized by reverse-phase HPLC fractionation followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom contained proteins belonging to 8 types of families. PI Zn(2+)-metalloproteinases and K49 PLA(2) molecules comprise over 65% of the venom proteins. Other venom protein families comprised PIII Zn(2+)-metalloproteinases (11.3%), D49 PLA(2)s (10.2%), l-amino acid oxidase (5.7%), the medium-sized disintegrin colombistatin (5.6%), serine proteinases (1%), bradykinin-potentiating peptides (0.8%), a DC-fragment (0.5%), and a CRISP protein (0.1%). A comparison of the venom proteomes of B. colombiensis and B. atrox did not support the suggested synonymy between these two species. The closest homologues to B. colombiensis venom proteins appeared to be toxins from B. asper. A rough estimation of the similarity between the venoms of B. colombiensis and B. asper indicated that these species share approximately 65-70% of their venom proteomes. The close kinship of B. colombiensis and B. asper points at the ancestor of B. colombiensis as the founding Central American B. asper ancestor. This finding may be relevant for reconstructing the natural history and cladogenesis of Bothrops. Further, the virtually indistinguishable immunological crossreactivity of a Venezuelan ABC antiserum (raised against a mixture of B. colombiensis and Crotalus durissus cumanensis venoms) and the Costa Rican ICP polyvalent antivenom (generated against a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) towards the venoms of B. colombiensis and B. asper, supports this view and suggests the possibility of indistinctly using these antivenoms for the management of snakebites by any of these Bothrops species. However, our analyses also evidenced the limited recognition capability or avidity of these antivenoms towards a number of B. colombiensis and B. asper venom components, most notably medium-size disintegrins, bradykinin-potentiating peptides, PLA(2) proteins, and PI Zn(2+)-metalloproteinases.