Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Origin and Characterization of Extracellular Vesicles Present in the Spider Venom of Ornithoctonus hainana.

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.

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach.

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 A2, 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.

Determining the Structures of the Snake and Spider Toxins by X-Rays.

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.

Samsum ant venom modulates the immune response and redox status at the acute toxic dose in vivo

Background:Ant venoms express surface molecules that participate in antigen presentation involving pro- and anti-inflammatory cytokines. This work aims to investigate the expression of MHC-II, CD80 and CD86 on the polymorphonuclear cells (PMNs) in rats injected with samsum ant venom (SAV).Methods:Rats were divided into three groups - control, SAV-treated (intraperitoneal route, 600 μg/kg), and SAV-treated (subcutaneous route, 600 μg/kg). After five doses, animals were euthanized and samples collected for analysis.Results:The subcutaneous SAV-trated rats presented decreased levels of glutathione with increased cholesterol and triglyceride levels. Intraperitoneal SAV-treated animals displayed significantly reduced concentrations of both IFN-γ and IL-17 in comparison with the control group. However, intraperitoneal and subcutaneous SAV-treated rats were able to upregulate the expressions of MHC-II, CD80 and CD86 on PMNs in comparison with the control respectively. The histological examination showed severe lymphocyte depletion in the splenic white pulp of the intraperitoneal SAV-injected rats.Conclusion:Stimulation of PMNs by SAV leads to upregulation of MHC-II, CD 80, and CD 86, which plays critical roles in antigen presentation and consequently proliferation of T-cells. Subcutaneous route was more efficient than intraperitoneal by elevating MHC-II, CD80 and CD86 expression, disturbing oxidative stability and increasing lipogram concentration.

ArachnoServer 3.0: an online resource for automated discovery, analysis and annotation of spider toxins.

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.

Seven novel modulators of the analgesic target NaV 1.7 uncovered using a high-throughput venom-based discovery approach.

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.

The history of venomous spider identification, venom extraction methods and antivenom production: a long journey at the Butantan Institute, São Paulo, Brazil

The article provides a historical report on venomous spider identification, venom obtainment methods and serum production at the Butantan Institute, São Paulo, Brazil. It is based on literature and personnal experience during the last 50 years. This result is the discovery that the real species causing potential severe human accidents were the spiders of the genus Loxosceles and Phoneutria.

Venomics of the spider Ornithoctonus huwena based on transcriptomic versus proteomic analysis.

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.

Determination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry of the extensive disulfide bonding in tarantula venom peptide Psalmopeotoxin I.

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.

Morphological characterization of the venom apparatus in the wolf spider Lycosa singoriensis (Laxmann, 1770)

The wolf spider Lycosa singoriensis (Laxmann, 1770) (Lycosidae: Araneae) is distributed throughout central and eastern Europe, including Russia, Kazakhistan and Turkey. This study describes the venom apparatus morphology of L. singoriensis through scanning electron microscopy (SEM). Its structure follows the general architecture observed in other spiders. Generally, a venom apparatus is composed by a pair of venom glands and chelicerae. L. singoriensis chelicerae are robust and consist of a stout basis and a movable apical segment (fang). The fang rests in a groove on the basal segment that is covered by different types of hair. L. singoriensis venom glands present equal size and measure about 4 mm in length. Each gland is enclosed by irregular muscular layers.(AU)

Proteomic analysis of Latrodectus tredecimguttatus venom for uncovering potential latrodectism-related proteins.

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.

Proteome and peptidome profiling of spider venoms.

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.

Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys jingzhao.

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.

Functional structure of Agelena labyrinthica's (Araneae:Agelenidae) venom gland and electrophoresis of venom.

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.

Sequence-specific assignment of 1H-NMR resonance and determination of the secondary structure of Jingzhaotoxin-I.

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.

Detection of Loxosceles species venom in dermal lesions: a comparison of 4 venom recovery methods.

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.

A new assay for the detection of Loxosceles species (brown recluse) spider venom.

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 main products of the low molecular mass fraction in the venom of the spider Latrodectus menavodi.

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.

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping.

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.