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Extracellular vesicles (EVs), including exosomes and microvesicles, are membranous vesicles released from nearly all cellular types. They contain various bioactive molecules, and their molecular composition varies depending on their cellular origin. As research into venomous animals has progressed, EVs have been discovered in the venom of snakes and parasitic wasps. Although vesicle secretion in spider venom glands has been observed, these secretory vesicles' origin and biological properties are unknown. In this study, the origin of the EVs from Ornithoctonus hainana venom was observed using transmission electron microscopy (TEM). The Ornithoctonus hainana venom extracellular vesicles (HN-EVs) were isolated and purified by density gradient centrifugation. HN-EVs possess classic membranous vesicles with a size distribution ranging from 50 to 150 nm and express the arthropod EV marker Tsp29Fb. The LC-MS/MS analysis identified a total of 150 proteins, which were divided into three groups according to their potential function: conservative vesicle transport-related proteins, virulence-related proteins, and other proteins of unknown function. Functionally, HN-EVs have hyaluronidase activity and inhibit the proliferation of human umbilical vein endothelial cells (HUVECs) by affecting the cytoskeleton and cell cycle. Overall, this study investigates the biological characteristics of HN-EVs for the first time and sheds new light on the envenomation process of spider venom.

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga . We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A 2 , kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Snake and spider venom is a complex mixture that contains proteins, peptides, and small organic and inorganic compounds. In contrast to spider venom, snake venom proteins are well known both functionally and structurally. This work describes methods for purification and crystallization of snake and spider venom toxins and their three-dimensional structure determination by X-ray crystallography.

Fingerprinting by means of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a tool for rapidly detecting proteinaceous compounds from spider venoms. Here we describe an optimized protocol and discuss methodological details with the aim of providing a platform for obtaining the most informative and reproducible mass spectral data.

In the last decades, main advances were achieved in the identification, structural and pharmacological characterization of Phoneutria nigriventer toxins. However, studies on the venom-producing apparatus are rare. Presently, we applied immunolabeling to historesin-embedded cross-sections of P. nigriventer venom glands. Toxins and toxin-secreting cells were successfully located in situ, using laser confocal scanning microscopy. The methodological strategy was successful and may be applied in future studies on venom glands and other secreting tissues, in general., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Diagnostic tests for arachnid accidents remain unavailable for patients and clinicians. Together with snakes, these accidents are still a global medical concern, and are recognized as neglected tropical issues. Due to arachnid toxins' fast mechanism of action, quick detection and quantification of venom is required to accelerate treatment decisions, rationalize therapy, and reduce costs and patient risks. This review aims to understand the current limitations for arachnid venom identification and quantification in biological samples. We benchmarked the already existing initiatives regarding test requirements (sample or biomarkers of choice), performances (time, detection limit, sensitivity and specificity) and their validation (on animal models or on samples from envenomed humans). Our analysis outlines unmet needs for improving diagnosis and consequently treatment of arachnid accidents. Hence, based on lessons from past attempts, we propose a road map for raising best practice guidelines, leading to recommendations for future progress in the development of arachnid diagnostic assays.

Summary: ArachnoServer is a manually curated database that consolidates information on the sequence, structure, function and pharmacology of spider-venom toxins. Although spider venoms are complex chemical arsenals, the primary constituents are small disulfide-bridged peptides that target neuronal ion channels and receptors. Due to their high potency and selectivity, these peptides have been developed as pharmacological tools, bioinsecticides and drug leads. A new version of ArachnoServer (v3.0) has been developed that includes a bioinformatics pipeline for automated detection and analysis of peptide toxin transcripts in assembled venom-gland transcriptomes. ArachnoServer v3.0 was updated with the latest sequence, structure and functional data, the search-by-mass feature has been enhanced, and toxin cards provide additional information about each mature toxin., Availability and Implementation: http://arachnoserver.org., Contact: support@arachnoserver.org., Supplementary Information: Supplementary data are available at Bioinformatics online.

The arms race between specialist predators and their prey has resulted in the evolution of a variety of specific adaptations. In venomous predators, this can include venom composition, particularly if predators are specialized on dangerous prey. Here, we performed an integrative study using six species of highly specialized ant-eating spiders of the genus Zodarion to investigate their phylogeny, realized trophic niche, efficacy in the capture of various ant species and venom composition. Data on natural diet obtained by next-generation sequencing and field observations showed that the six Zodarion species exploit different ant species. Their phylogeny, based on mitochondrial and nuclear genes, correlated with the composition of their natural prey, indicating that closely related Zodarion species specialize on similar ant species. Prey-capture parameters differed among Zodarion species suggesting prey-specific efficacy. Similarly, the venom profiles of both low and high molecular compounds differed among species. Only the profiles of low molecular compounds were correlated with capture efficacy parameters, suggesting that the venom of Zodarion spiders contains prey-specific components. Our study suggests that Iberian Zodarion spiders are specialized on particular ant species., (© 2018 John Wiley & Sons Ltd.)

Background: Pardosa pseudoannulata is a prevailing spider species, and has been regarded as an important bio-control agent of insect pests in farmland of China. However, the available genomic and transcriptomic databases of P. pseudoannulata and their venom are limited, which severely hampers functional genomic analysis of P. pseudoannulata. Recently high-throughput sequencing technology has been proved to be an efficient tool for profiling the transcriptome of relevant non-target organisms exposed to Bacillus thuringiensis (Bt) protein through food webs., Results: In this study, the transcriptome of the venom apparatus was analyzed. A total of 113,358 non-redundant unigenes were yielded, among which 34,041 unigenes with complete or various length encoding regions were assigned biological function annotations and annotated with gene ontology and karyotic orthologous group terms. In addition, 3726 unigenes involved in response to stimulus and 720 unigenes associated with immune-response pathways were identified. Furthermore, we investigated transcriptomic changes in the venom apparatus using tag-based DGE technique. A total of 1724 differentially expressed genes (DEGs) were detected, while 75 and 372 DEGs were functionally annotated with KEGG pathways and GO terms, respectively. qPCR analyses were performed to verify the DEGs directly or indirectly related to immune and stress responses, including genes encoding heat shock protein, toll-like receptor, GST and NADH dehydrogenase., Conclusion: This is the first study conducted to specifically investigate the venom apparatus of P. pseudoannulata in response to Bt protein exposure through tritrophic chain. A substantial fraction of transcript sequences was generated by high-throughput sequencing of the venom apparatus of P. pseudoannulata. Then a comparative transcriptome analysis showing a large number of candidate genes involved in immune response were identified by the tag-based DGE technology. This transcriptome dataset will provide a comprehensive sequence resource for furture molecular genetic research of the venom apparatus of P. pseudoannulata.

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little research has been carried out on the venom of Australian tarantulas. We therefore investigated the venom profile of the Australian theraphosid spider Phlogius crassipes and examined whether there are ontogenetic changes in venom composition. Spiders were divided into four ontogenic groups according to cephalothorax length, then the venom composition of each group was examined using gel electrophoresis and mass spectrometry. We found that the venom of P. crassipes changes continuously during development and throughout adulthood. Our data highlight the need to investigate the venom of organisms over the course of their lives to uncover and understand the changing functions of venom and the full range of toxins expressed. This in turn should lead to a deeper understanding of the organism's ecology and enhance the potential for biodiscovery.

Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Context: Funnel-web spider (Atrax and Hadronyche spp.) envenoming is rare but causes severe neuromuscular, autonomic, and cardiac effects. A rabbit-derived IgG antivenom is available, but venom detection in patients has not been reported., Objective: To use serial venom and antivenom concentrations to better define envenoming and antivenom effectiveness., Materials and Methods: Serum was collected from nine patients with suspected funnel-web spider bites and clinical effects were recorded. Venom-specific enzyme immunoassays were developed to measure funnel-web spider venom and antivenom concentrations. Goat anti-rabbit whole serum was coupled to UltraLink resin and added to samples to remove bound venom and measure free venom. Antivenom efficacy was defined as antivenom binding all free venom and antivenom effectiveness as resolution of clinical features., Results: Venom was detectable in samples from six of nine patients. In three patients without venom detected, there were only moderate effects, which did not completely respond to antivenom in all cases and no spider was identified. In five of six cases, a male Atrax spp. (Sydney funnel-web) spider was identified. Three patients had moderate envenoming which responded to antivenom. Three patients had severe envenoming and developed catecholamine-induced myocarditis and acute pulmonary oedema. Although cholinergic and non-specific clinical features appeared to respond to antivenom, myocarditis and pulmonary oedema lasted 2-4 days. Median venom concentration pre-antivenom in five patients with samples was 5.6 ng/ml (3-35 ng/ml), and immediately post-antivenom decreased to a median of 0 ng/ml (0-1.8 ng/ml). Post-antivenom venom concentrations decreased when bound venom was removed; median, 0 ng/ml (0-0.9 ng/ml), indicating that most venom detected post-antivenom was bound. There was recurrence of venom and clinical features in one patient when a pressure bandage was removed., Conclusions: Detection of venom in suspected funnel-web spider bites identified definite cases with characteristic envenoming and a spider was identified. Measurement of venom concentrations pre- and post-antivenom demonstrated that venom was bound by antivenom, but in severe cases cardiac toxicity was not reversed.

Background and Purpose: Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel NaV 1.7 (hNaV 1.7) are indifferent to pain, making hNaV 1.7 a promising target for analgesic development. Since spider venoms are replete with NaV channel modulators, we examined their potential as a source of hNaV 1.7 inhibitors., Experimental Approach: We developed a high-throughput fluorescent-based assay to screen spider venoms against hNaV 1.7 and isolate 'hit' peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNaV 1.7 was transplanted into the homotetrameric KV 2.1 channel., Key Results: We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNaV 1.7. By deconvoluting 'hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNaV 1.7 with a high level of selectivity over all other subtypes, except hNaV 1.1. We showed that Hd1a is a gating modifier that inhibits hNaV 1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability., Conclusion and Implications: Our data indicate that spider venoms are a rich natural source of hNaV 1.7 inhibitors that might be useful leads for the development of novel analgesics., (© 2015 The British Pharmacological Society.)

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

1. Disruptive natural selection resulting from specialization on different hosts is recognized as one of the most important driving forces in the diversification of herbivores and parasites. It has been proposed that a similar mechanism could apply to carnivorous predators too, although the evidence is still lacking. 2. Here, we show that the differentiation of biotypes of specialized ant-eating spiders of the genus Zodarion has probably been induced by prey-shifting. We focused on two forms of one species Z. styliferum from the Iberian Peninsula that presumably represent ecological races. We conducted geographic, ecological, venom-oriented, reproductive and genetic divergence analysis among multiple populations collected at a number of sites across Portugal and Madeira. 3. Geographic analysis revealed that the two forms occur in mosaic sympatry. Each form was found to associate in nature with a different ant species in a different habitat. Specifically, the styliferum form hunted predominantly Messor ants, and the extraneum form hunted mainly Camponotus ants. Laboratory experiments revealed that the two forms exhibit a significant preference for attacking focal ants, demonstrating higher paralysis efficiency, and also show different venom composition. Cross-mating of the two forms was significantly less likely than between pairs of the same form, suggesting moderate assortative mating. Phylogenetic analyses indicate low genetic differentiation of the two forms and parallel-repeated evolution of biotypes. 4. Adaptive prey-shifting correlated with habitat preference are at present the most valid explanations for biotype formation in Zodarion. The speciation of ant-eating Zodarion spiders thus appears to follow a scenario similar to that of host-shifting in parasites and herbivores., (© 2012 The Authors. Journal of Animal Ecology © 2012 British Ecological Society.)

The venom of Loxosceles spiders produces severe dermonecrotic damage, intravascular hemolysis, systemic alterations and risk of death. Clostridium perfringens is present in the microbial flora of the fangs and venom glands of Loxosceles intermedia. Its inoculation with the venom may infect the wound site and exacerbate the dermonecrotic damage. This anaerobic bacterium is widely distributed in nature and capable of damage with similar characteristics and severity to the spider venom. In this study we isolated and characterized species of Clostridium from the fangs and venom glands of Loxosceles laeta, including C. perfringens. The sensitivity patterns of different isolates of C. perfringens were evaluated by minimum inhibitory concentration against penicillin, ampicillin, erythromycin, gentamicin, chloramphenicol, clindamycin and tetracycline, under anaerobic conditions, using the method of microdilution in broth. Strain C. perfringens H28 showed resistance to penicillin, ampicillin, tetracycline and chloramphenicol. Resistance to penicillin and ampicillin was mediated by beta-lactamase. In vivo evaluation of dermonecrosis in rabbits using L. laeta venom co-inoculated with isolate C. perfringens H28 produced an increase in the area of dermonecrotic lesions in the presence of penicillin and tetracycline, but not with gentamicin. Antibiotic therapy Loxosceles poisoning should be re-evaluated, considering the existence of multi-resistant strains of C. perfringens., ((c) 2010. Published by Elsevier Ltd.)

Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The spider Ornithoctonus huwena is a venomous spider found in southern China. Its venom is a complex mixture of numerous biologically active components. In this study, 41 novel unique transcripts encoding cellular proteins or other possible venom components were generated from the previously constructed cDNA library. These proteins were also annotated by KOG (eukaryotic orthologous group) and GO (gene ontology) terms. A novel cellular transcript contig encoding an EF-hand protein (named HWEFHP1) was found, which might be involved in the secretion of toxins in the venom glands. In order to have an overview of the molecular diversity of the O. huwena venom, the datasets of all the transcripts, peptides and proteins known so far were analyzed. A comparison of the data obtained through a proteomic versus a transcriptomic approach, revealed that only 15 putative cystine knot toxins (CKTs) were identified by both approaches, 29 transcripts coding for CKTs were found in the transcriptome but not as translated peptides in the venom proteome. However, no cellular protein with identical molecular weight was identified by both approaches. Our data may contribute to a deeper understanding of the biology and ecology of O. huwena and the relationship between structure and function of individual toxins., (2010 Elsevier Inc. All rights reserved.)

We report here a case of systemic loxoscelism, confirmed by bite-site skin surface swab. Features of systemic loxoscelism present in this case included debilitating symptoms, a classic local bite-site reaction, hemolysis causing loss of approximately 15% of the blood volume within 72 hours, and a symptomatic exanthem. A skin surface ELISA test was used to confirm the presence of venom. This test enables confirmation of cases of loxoscelism for which no spider is found.

Background: Diagnosis of Loxosceles reclusa envenomations is currently based upon clinical presentation. An enzyme-linked immunosorbent assay (ELISA) can detect surface Loxosceles venom at the envenomation site, allowing diagnostic confirmation. The length of time that venom on the skin is recoverable non-invasively is unknown., Materials and Methods: To investigate duration of recoverable venom antigen, whole venom and fractionated sphingomyelinase D venom aliquots were injected subcutaneously in New Zealand White rabbits. Cotton and Dacron swabs were compared for venom recovery over a 21-day period using a surface swab technique., Results: Significant amounts of Loxosceles reclusa antigen were found on the surface of rabbit skin after experimental injection of whole venom and sphingomyelinase D. The duration of recoverable antigen using this experimental model appears to be at least two weeks and as long as 21 days in some cases., Conclusions: Because the duration of the recoverable antigen is seen to be at least two weeks, the ELISA venom test appears capable of detecting venom on most patients presenting with Loxosceles envenomations. This detection system will allow the physician more accurate determination of whether the lesion is from a brown recluse spider or some other agent that can cause this type of necrotic ulcer.

This communication revises the state of the art concerning antivenoms against snakes, spiders and scorpions. An overview of the historical facts that preceded the therapeutic use of antibodies is mentioned. A brief list of the major protein components of these venomous animals is revised with a short discussion of what is known on the proteomic analysis of their venoms, but the emphasis is placed on the type of antivenoms available commercially, including pertinent literature and addresses of the companies that prepare these antivenoms. The final section revises and discusses current research on the field and new potential applications that are being developed geared at obtaining new therapeutic antibodies or fragments of antibodies for neutralization of toxic components of venomous animals.

Loxosceles intermedia spider venom was subjected to proteomic analysis through a MudPIT shot-gun approach to identify the protein composition. Were identified 39 proteins which seem to responsible by the lesion of different types of tissues, to some physiopathological actions and by the prevention of structural damage to the toxin structures.

Psalmopeotoxin I (PcFK1) is a 33-residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. This peptide specifically inhibits the intra-erythrocyte stage of Plasmodium falciparum in vitro. It contains six cysteine residues forming three disulfide bridges and belongs to the superfamily of natural peptides containing the inhibitor cystine knot (ICK) fold. We produced the wild-type and mutated forms of the recombinant peptide to examine the mechanism of action of PcFK1. The purified toxins were consistently produced as two isobaric peptides (r-PcFK1-1 and r-PcFK1-2) with different retention properties but identical anti-plasmodial -biological activity. Comparison of (15)N-NMR heteronuclear single quantum correlation spectra revealed that although rPcFK1-1 was highly structured, rPcFK1-2 does not have a stable three-dimensional structure. We used high-energy collision-induced fragmentation of the peptides with a matrix-assisted laser desorption/ionization tandem time-of- flight mass spectrometer to further investigate the structure of the native peptides in its natural form and produced in E. coli. The fragmentation spectra of the native peptides were very complex due to the occurrence in the spectrum of ions resulting from (1) cross-linking of fragments through a disulfide bridge and (2) asymmetric fragmentations of the disulfide bridges and (3) multiple neutral losses. The tandem mass spectrometry fragmentation pattern of r-PcFK1-1 was similar to that of the natural peptide isolated from crude venom, but r-PcFK1-2 had a clearly distinct fragmentation pattern, more closely resembling the fragmentation spectra of reduced and alkylated peptides. Observed ions could be attributed to specific fragments by comparing spectra between the wild-type and selected variants with point mutations (Y11W, R20T, Y26W, K28V). The disulfide connections in r-PcFK1-2 differed from those of the native peptide and showed a rare disulfide bridge between vicinal cysteine residues. The r-PcFK1_(R20T) variant showed a very limited fragmentation pattern when analyzed in positive mode but displayed much more fragmentation in negative mode pointing out the importance of the R20 residue in the fragmentation of PcFK1. Using the reductive matrix 1,5-diaminonaphtalene promoted strongly in source decay fragmentation of the peptides in MS mode. Our findings illustrated the critical role of the electronic environment around the central Cys(18)-Cys(19) doublet in PcFK1 in internal fragmentation of the peptide.

Spider venoms are an important source of novel molecules with different pharmacological properties. Recent technological developments of proteomics, especially mass spectrometry, have greatly promoted the systematic analysis of spider venom. The enormous diversity of venom components between spider species and the lack of complete genome sequence, and the limited database of protein and peptide sequences make spider venom profiling a challenging task and special considerations for technical strategies are required. This review highlights recently used methods for spider venom profiling. In general, spider venom profiling can be achieved in two parts: proteome profiling of the components with molecular weights above 10 kDa, and peptidome profiling of the components with a molecular weight of 10 kDa or under through the use of different methods. Venom proteomes are rich in various enzymes, hemocyanins, toxin-like proteins and many unknown proteins. Peptidomes are dominated by peptides with a mass of 3-6 kDa with three to five disulfide bonds. Although there are some similarities in peptide superfamily types of venoms from different spider species, the venom profile of each species is unique. The linkage of the peptidomic data with that of the cDNA approach is discussed briefly. Future challenges and perspectives are also highlighted in this review.

Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.

Purpose: The authors report a case of Loxosceles reclusa infestation on the eyelid., Methods: The specimen collected by swabbing the lesions with gauze was tested by using a venom-specific enzyme-linked immunosorbent assay. Loxosceles venom was detected in specimen. The patient was managed with conservative therapy that included saline compresses and ocular lubrication., Results: The case presented with severe edema and necrosis on the left upper eyelid. On laboratory examination, a significant left shift of white blood cell count was detected. Although residual scar of the eyelid and punctate epitheliopathy on the inferior of cornea was detected, vision was not impaired., Conclusions: Supportive therapy may be reasonable treatment for Loxosceles reclusa infestation on the eyelids. The presence of venom proteins detected with an enzyme-linked immunosorbent assay technique is beneficial for supporting the diagnosis of Loxosceles envenomatio.

Animal venoms and toxins are now recognized as major sources of bioactive molecules that may be tomorrow's new drug leads. Their complexity and their potential as drug sources have been demonstrated by application of modern analytical technologies, which have revealed venoms to be vast peptide combinatorial libraries. Structural as well as pharmacological diversity is immense, and mass spectrometry is now one of the major investigative tools for the structural investigation of venom components. Recent advances in its use in the study of venom and toxins are reviewed. The application of mass spectrometry techniques to peptide toxin sequence determination by de novo sequencing is discussed in detail, in the light of the search for novel analgesic drugs. We also present the combined application of LC-MALDI separation with mass fingerprinting and ISD fragmentation for the determination of structural and pharmacological classes of peptides in complex spider venoms. This approach now serves as the basis for the full investigation of complex spider venom proteomes, in combination with cDNA analysis., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Chinese tarantula, Chilobrachys jingzhao is one of the most venomous spiders in southern China and its venom is a mixture of various compounds with diversified biological activities. The proteome of C. jingzhao venom was analyzed by proteomic techniques. Proteins with molecular weight of over 10 kDa, indicated by gel-filtration and SDS-PAGE, were analyzed using 2-DE and MALDI-TOF/TOF and LC/ESI-Q-TOF MS. More than 90 proteins were detected, with 47 confirmed by sequence similarity search using mass spectrum driven basic local alignment search tool (MS BLAST). On the other hand, peptides with MW lower than 10 kDa were separated by HPLC and identified by MALDI-TOF MS and Edman degradation sequencing. About 120 peptides were detected, 60 of which were fully or partially sequenced. Our results indicate that peptides with MW lower than 10 kDa are the major components in the crude venom of C. jingzhao. Like those of other tarantulas, these peptides are very likely to act on various ion channels. These results pave a way for further detailed structure-function correlation analysis of the individual toxins present in the venom of C. jingzhao.

Background: Confirmation of mild bites caused by Loxosceles reclusa with swab testing has not been previously documented, to our knowledge., Methods: We report a case using an enzyme-linked immunosorbent assay (ELISA) test., Results: A lesion lacking necrosis or other specific signs of loxoscelism was confirmed by identification of the Loxosceles venom and further confirmed by identification of a spider found in the patient's bed., Limitations: This is a pilot single-case report for this enzyme-linked immunosorbent assay test., Conclusions: A sensitive and specific enzyme-linked immunosorbent assay designed to detect Loxosceles venom, using a specimen obtained by swabbing the lesion, can aid in diagnosis of loxoscelism.

In order to establish a venom fingerprint and a peptide profile of the Lasiodora parahybana tarantula venom gland, we used conventional methods such as reversed phase liquid chromatography coupled to an electrospray-ionisation hybrid quadrupole time of flight mass spectrometer (LC/ESI-QqTOFMS), matrix-assisted laser desorption/ionization time-of-flight-MS (MALDI-TOFMS) and direct study of L. parahybana venom by nanospray-ionization QqTOFMS (nanoESI-QqTOFMS) and a new technology for the direct analysis of fresh tissues using MALDI-TOFMS. The analysis of the crude venom allowed the characterization of specific juvenile and adult biomarkers. In situ MALDI analysis of L. parahybana venom gland sections revealed different peptide expression levels all along the gland and non-processed peptide precursors, demonstrating the power of the method for the dynamic investigation of peptide evolution in the venom gland of spiders.

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification.

The funnel-web spider, Agelena labyrinthica, is widely distributed throughout Turkey. The objective of the present study was to describe the histological and functional fine structure of A. labyrinthica's venom gland by using light microscope, scanning (SEM) and transmission electron microscope (TEM). We have also preliminarily analyzed venom components by SDS-PAGE. Each venom gland has surrounded by a thin adventitia and gross striated muscular bundles. Basal lamina underlies between muscular bundles and the inner glandular epithelium, and ties up them each other. The striated muscular bundles spirally covered venom gland has been observed by SEM. Intricate relations formed between motor neuron axons and the muscle fibers have been revealed by TEM. The secretory epithelium, which made up of simple columnar cells, formed the secretory region of the venom gland. The secretory surface of the gland was increased by a sort of fringes extended from basal membrane into the gland lumen. The epithelial cells have many rough endoplasmic reticulum, mitochondria and different size and shape of secretory granules. These granules have been accumulated in apical portion of the secretory cells. After the gland is emptied, the apical portions of secretory cells deteriorate and the basal epithelial cells regenerate the columnar cells. The analysis of A. labyrinthica venom, which was achieved by SDS-PAGE showed that there have been at least seven components ranging from 10 to 40 kDa molecular weight.

Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by d alphaN, d alphadelta, d betaN and d NN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.

Mass spectrometry is a technique for the analysis and very sensitive identification of molecules. It allows one to determine the mass of the studied product, whether pure or in a mixture, and provides some information on its molecular structure. In the particular case of peptides, this method can, under certain conditions, also provide information on the amino acid sequence. There are two complementary methods in mass spectrometry for the study of the biological molecules: i) ionisation by laser desorption assisted by matrix (MALDI) coupled to a mass analyser of the time of flight type (TOF), which is very effective for the direct study of a mixture of products and ii) ionisation by electronebulisation (ESI) coupled to mass analysers of the quadripolar type and time of flight (Qq-TOF), which allows the interfacing between high phase liquid chromatography and mass spectrometry. These two complementary techniques were already used to draw up toxin charts of snake and spider venoms. The purpose is to be able to characterise species based on an actual peptide print of poisonous gland secretions.

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.

The spider Cupiennius salei needs 0.01-10 microl venom to kill a prey item. Since its venom glands contain only 10 microl and regeneration requires 8-16 days C. salei should use its venom very economically. By a monoclonal antibody we measured, for the first time, the amounts of venom injected by a spider into different prey types. Crickets and stick insects, as victims without special defence mechanism, received only the minimum amount of venom which is not significantly different from the LD(50). Blowflies and ground beetles received considerably more venom because they are difficult to overwhelm or even endanger the spider by their defence behaviour. These results support our venom optimisation hypothesis which supposes that spiders use their venom as economically as possible., (Copright 2002 Elsevier Science Ltd.)

Study Objective: Loxosceles species spider envenomations may produce necrotic, disfiguring dermal inflammatory lesions resembling neutrophilic dermatoses. With definitive treatment options lacking, clinicians are reluctant to obtain invasive biopsy specimens for diagnostic analysis. We compared less invasive venom collection methods and determined the time limit after inoculation for feasible venom recovery in an animal model., Methods: Nine New Zealand rabbits were randomized to 1 of 3 groups (n=3). Groups 1 and 2 were inoculated intradermally with 3 microg of L reclusa venom at 5 inoculation sites per rabbit. Albumin (3 microg) was injected intradermally in each rabbit as a negative control. Hair (group 1) and aspirate samples (group 2) were collected (1 time per site) over a 1-week period after inoculation. Group 3 was inoculated with 3 microg of Loxosceles species venom on 1 flank and 3 microg of albumin on the opposite flank. Daily serum specimens were collected over a 7-day period. On day 7, dermal punch biopsy specimens were taken from the venom and control inoculation sites. Hair, aspirate, biopsy, and serum specimens were assayed for venom by using an enzyme-linked immunosorbent assay. A generalized linear model was fit with the generalized estimating equation method to estimate the mean differences between groups., Results: Venom was detected in hair, aspirate, and biopsy specimens on all days of the study period. Hair samples yielded venom recovery on day 1 (median 0.062 ng/100 microL; mean difference 0.054 ng/100 microL; 95% confidence interval [CI] 0.048 to 0.059) through day 7 (median 0.020 ng/100 microL; mean difference 0.020 ng/100 microL; 95% CI 0.013 to 0.027). Aspirates were positive for venom recovery on day 1 (median 0.275 ng/100 microL; mean difference 0.231 ng/100 microL; 95% CI 0.192 to 0.271) through day 7 (median 0.0 ng/100 microL; mean difference 0.032 ng/100 microL; 95% CI -0.18 to 0.078). The highest venom yield was from the biopsy specimens (median 1.75 ng/100 microL; mean difference 0.041 ng/100 microL; 95% CI 0.033 to 0.027). Venom was undetectable in all serum samples., Conclusion: Loxosceles species venom is detectable in hair, aspirate, and dermal biopsy specimens at least 7 days after venom inoculation and undetectable in serum by using the rabbit model.

Study Objective: Dermal lesions from unrelated arthropod species and medical causes appear similar to Loxosceles species (brown recluse spider) bites. This may result in delayed diagnosis and treatment. We developed a sensitive Loxosceles species venom enzyme-linked immunosorbent assay (ELISA) and characterized the specificity of the assay by evaluating antigenic cross-reactivity from a variety of North American arthropod venoms., Methods: North American arthropod (14 spiders, 2 scorpions, and 1 bee) venoms were studied. Three venom amounts (diluted in 100 microL of ELISA buffer) were assayed: 16,000 ng, 2,000 ng, and 40 ng. The latter quantity was selected because this is the observed maximum amount of venom we detect when inoculating dermis with amounts likely to be deposited by a spider bite. The larger venom amounts are overwhelming quantities designed to test the limits of the assay for arthropod venom cross-reactivity. Similar amounts of Loxosceles species venom and bovine albumin served as positive and negative controls, respectively., Results: At the lowest amount of venom tested (40 ng), the ELISA detected only the Loxosceles species positive control. When 2,000 ng was assayed, only Scytodes fusca and Kukulcania hibernalis arachnid venoms (in addition to Loxosceles species) cross-reacted to the assay. Finally, at 16,000 ng, the ELISA assay modestly detected Diguetia canities, Heteropoda venatoria, Tegenaria agrestis, Plectreurys tristes, Dolomedes tenebrosus, and Hadrurus arizonensis arachnid venoms., Conclusion: Cross-reactivity was observed in 8 of 17 North American arthropod venoms when large venom amounts were assayed with a Loxosceles species ELISA. By using a relevant quantity of venom, 40 ng, the assay was specific for Loxosceles species venom. The venom specificity of the ELISA may allow clinical application in Loxosceles species endemic regions of North America.

The venom of the brown spider is remarkable because it causes dermonecrotic injury, hemorrhagic problems, hemolysis, platelet aggregation and renal failure. The mechanism by which the venom causes hemorrhagic disorders is poorly understood. Rabbits intradermally exposed to the venom showed a local hemorrhage starting 1 h after inoculation and reaching maximum activity between 2 and 3 days. Biopsies examined by light and transmission electron microscopy showed subendothelial blebs, vacuoles and endothelial cell membrane degeneration in blood vessels, plasma exudation into connective tissue, and fibrin and thrombus formation within blood vessels. Loxosceles intermedia venom incubated with fibrinogen partially degrades Aalpha and Bbeta chains of intact fibrinogen, and significantly cleaves all Aalpha, Bbeta and gamma chains when they were separated or when fibrinogen is denatured by boiling. Proteolytic kinetic studies showed that the Aalpha chain is more susceptible to venom hydrolysis than the Bbeta chain. The fibrinogenolysis is blocked by ethylenediamine tetraacetic acid and 1,10-phenanthroline, but not by other protease inhibitors. Human plasma incubated with the venom had coagulation parameters such as prothrombin time, activated partial thromboplastin time and thrombin time increased. Through molecular sieve chromatography, we isolated a venom toxin of 30 kDa with fibrinogenolytic activity. We propose that the local and systemic hemorrhagic disorders evoked in loxoscelism are consequences of direct venom fibrinogenolysis together with cytotoxicity to subendothelial structures and endothelial cells in blood vessels.

Animal venoms are important sources of novel pharmacological tools, useful in biochemical characterization of their receptors. Venom quality control, batch-to-batch homogeneity and high reproducibility of venom fractionation and toxin purification are crucial issues for biochemical and pharmacological studies. To address these issues, a study of the variability of tarantula spider venom samples was undertaken. Venom profiles of samples collected from individuals of different age and sex, and from sibling spiders of the same species, were generated by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and analyzed to assess venom variability and method accuracy. Sex-linked venom variation was studied on eight species. Clear qualitative differences were observed for six out of eight species, as well as quantitative differences. Age-related variation studied in Poecilotheria rufilata showed essentially age-related quantitative differences between adults of both sexes and immature juveniles. The venoms of nine siblings and three wild-collected Pterinochilus murinus were studied for individual variation, showing only very minor quantitative differences. On the same samples, the quality of MALDI-TOFMS venom fingerprinting was demonstrated to be highly reproducible. Our results show that tarantula venom peptide fingerprinting is a highly reliable identification method, that pooled batches of venom from several animals can be used for venom purification, that venom composition does not appear to be qualitatively related to ontogenesis in the spiders studied, and that qualitative sex-linked variation occurs across most species and may be important in activity studies., (Copyright 2002 John Wiley & Sons, Ltd.)

The low molecular mass fraction of the venom of the spider Latrodectus menavodi, a black widow spider domiciled in Madagascar, was analyzed with HPLC combined with electrospray mass spectrometry as lyophilized powder and as freshly milked liquid venom. The main components were found to be the purine derivatives adenosine (1), guanosine (2), inosine (3), and 2,4,6-trihydroxypurine (4). Compounds 1-3 are known as components of spider or snake venoms, but they have never been found previously in the venom of the species Latrodectus, whereas compound 4 has not previously been found in any spider venoms. The lyophilized and unlyophilized venoms were found to have identical composition.

Study Objectives: Widow spiders of the genus Latrodectus are found worldwide and produce similar clinical envenomation syndromes. In Australia, red-back spider antivenom (RBS-AV) is effective therapy for Latrodectus hasselti envenomation and it has been reported to reverse envenomation by other widow spiders. This study assessed the efficacy of RBS-AV in preventing in vitro and in vivo toxicity of widow spider venoms of North America and Europe., Methods: The binding of RBS-AV to alpha-latrotoxin and Latrodectus venoms (Latrodectus spp mactans, hesperus, lugubris, tredecimguttatus, hasselti) was assayed using Western blotting. Prevention of in vitro toxicity to alpha-latrotoxin and the same venoms was tested by pretreating an isolated chick biventer cervicis nerve-muscle preparation with RBS-AV. Prevention of in vivo toxicity was determined by a lethality study in male Balb/c mice (2.5 to 5x median lethal dose [LD50]) or alpha-latrotoxin (10x LD50) preincubated with antivenom or without RBS-AV (control)., Results: In Western blots, RBS-AV bound to alpha-latrotoxin and similar widow spider proteins in all venoms tested, indicating antigenic similarity with proteins found in RBS venom. Antivenom prevented the typical in vitro muscle contracture and loss of twitch tension seen with alpha-latrotoxin and the venoms tested. Control mice rapidly developed signs of envenomation, but mice treated with RBS-AV remained free of signs of envenomation., Conclusion: RBS-AV prevented both in vitro and in vivo toxicity from Latrodectus venoms and alpha-latrotoxin in mice. These data suggest that RBS-AV may be clinically effective in the treatment of envenomation resulting from the bite of other widow spiders.

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3-10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight >10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.

Fluorescein and horseradish peroxidase-labeled monoclonal antibodies were used to localize the predominant toxic peptide CSTX-1 in the venom gland of the spider Cupiennius salei. There was no polarity of CSTX-1 expression in repleted glands, whereas the glands of previously milked spiders showed a decreasing immunofluorescent response from the distal to the proximal portion. Detailed investigation revealed a new structure in the venom-secreting epithelium, which is postulated to be an evolutionary adaptation to increasing gland volume. CSTX-1 was found to be synthesized and stored as a fully active toxin within complex units, composed of long interdigitating cells running perpendicular to the muscular sheath and extending into the central lumen of the gland. These venom-producing units were found in all sectors of the gland, including the transitional region between the main gland and the venom duct. The venom is liberated from the venom-producing units into the glandular lumen following the contraction of the surrounding muscle layer. Free nuclei or other cellular fragments, which would have provided evidence for a holocrine secretion process, were not found in the glandular lumen or in the crude venom obtained by electrical stimulation. The fine regulation of the spider's venom injection process is postulated to be the function of the bulbous ampulla, situated in the anterior third of the venom duct.

The variation in venom dose with prey size of the neotropical wandering spider Cupieinnius salei was examined experimentally. Monoclonal antibodies were raised against the venom toxins of C. salei. Mab 9H3, recognizing the main toxin CSTX-1, was used to quantify the venom by enzyme-linked immunosorbent assay (ELISA). Crickets (Achta domesticus) in four size classes were randomly offered to sixteen mature female spiders at 14d intervals. The prey items were removed from spiders five minutes after the initial bite and subsequently homogenized for ELISA measurements. The quantity of venom expended was significantly related to the size of prey, ranging from 0.15 microl for the smallest (100 110 mg) to 1.53 microl for the largest (600-660 mg) crickets. Adaptations to prey size were also reflected in capturing behavior. None of the smallest, but almost 50% of the largest crickets were wrapped in silk following the spiders bite. Some other behavioral features may reduce the energetic costs of venom production. In 22% of the smallest crickets no venom was detectable, with the majority showing mechanical damage as a result of fang contact. This indicates. that C. salei does not rely exclusively on its venom when feeding on small prey. Some other aspects such as the site of the bite and the speed of paralyzation and their consequences associated with the amount of venom expended are discussed.

By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inability to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving action whereas serine-protease inhibitors, thiol-protease inhibitor and acid-protease inhibitor showed little or no effect on the proteolytic activity of the venom indicating involvement of a metalloproteinase. Zymogram analysis of venom detected a 35 kDa molecule with gelatinolytic activity. The metalloproteinase nature was further supported by its sensitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decreased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose affinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogenolytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,10 phenanthroline and APMA sensitivity on this proteolytic effect confirmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. intermedia venom with different specificities one gelatinolytic and another, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom.

The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose alpha 1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.

Spiders belonging to the genera, Nephila, Nephilengys, Araneus, etc., possess various polyamine toxins in their venom glands which paralyze insects by blocking the nerve-muscle signal transduction of glutaminergic synapses. More than 50 kinds of polyamine toxin analogs have been characterized which consist, in general, of the aromatic or heteroaromatic moiety connected with the combination of various types of polyamine chains. We are developing a new analytical system by means of a modern mass spectrometric technique which satisfies more rapid, highly sensitive and simple clean-up procedure without complicated chromatographic treatment. The trial of such approach has been performed by employing crude spider venom as the model material. This review article concerns with such proceeding of mass spectrometric analysis by microcolumn HPLC hyphenated FAB-MS system, continuous flow FAB-MS/MS system with high energy collision charge-remote fragmentation, solid and liquid matrix assisted laser desorption ionization (MALDI- and liquid MALDI-) technique connected with tandem mass spectrometer performing in the institute and in addition, femto-molar characterization of new spider venom spider polyamine toxins as a result of the application of such new techniques is addressed.

Spider venom glands store various novel neurotoxic acylpolyamines which present potent and irreversible inhibition of the glutamatergic synapses. We have developed a new highly sensitive analytical method to detect and characterize structures of those neurotoxins stored in a single venom gland using micro bore column high performance liquid chromatography (HPLC)-continuous flow FRIT fast atom bombardment mass spectrometry (HPLC-FAB/MS) and tandem mass spectrometry (MS/MS) array detection system. The high-energy collision induced dissociation (CID) spectra of sodium cationized spider toxins produced very strong structurally informative product ions which afforded the location of nitrogen atoms and the connectivity of methylene units within the polyamines. This methodology permitted detection of 40 amino acid containing acylpolyamines in the venom of Nephilengys borbonica from which structures of only five compounds were previously known.