Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom.

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

Oligopeptides analysis in spiderhawk's venom (Pepsis decorata Perty, 1833, Hymenoptera: Pompilidae)

Wasps have been neglected in toxinological studies, even with their diversity of species, when compared to other groups of venomous animals such as snakes, scorpions, and spiders. Solitary wasps, such as Pepsis decorata, are known for their mechanism of total or temporary paralysis of the host. In addition, their venoms are considered sources for studies of small peptides, bioactive peptides with neural and antimicrobial activities. In this work, some oligopeptides were analyzed by de novo sequencing identifying 39 oligopeptide sequences. Some sequences were similar to proctolin, a bradykinin-potentiating peptide, and poneritoxin, one bradykinin-related peptide. As proctolin-like peptides were the major constituent in distinct experimental conditions, it was selected for further in silico studies in order to understand its possible importance as a constituent of wasp venom and whether these peptides could be of biotechnological importance. We investigate its binding mode comparing with proctolin and we further analyzed the importance of the tyrosine-leucine-glutamic acid (YLE) tripeptide-motif conservation. This experimental, an in silico approach, increased the range of compounds identified in peptide analyses proving good characterization of little-known peptidic compounds.

Milk provisioning in oviparous caecilian amphibians

Among vertebrates, the yolk is commonly the only form of nutritional investment offered by the female to the embryo. Some species, however, have developed parental care behaviors associated with specialized food provisioning essential for offspring survival, such as the production of lipidic-rich parental milk in mammals. Here, we show that females of the egg-laying caecilian amphibian Siphonops annulatus provide similarly lipid-rich milk to altricial hatchlings during parental care. We observed that for 2 months, S. annulatus babies ingested milk released through the maternal vent seemingly in response to tactile and acoustic stimulation by the babies. The milk, composed mainly of lipids and carbohydrates, originates from the maternal oviduct epithelium’s hypertrophied glands. Our data suggest lactation in this oviparous nonmammalian species and expand the knowledge of parental care and communication in caecilians.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1.

Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

Spatial Distribution and Biochemical Characterization of Serine Peptidase Inhibitors in the Venom of the Brazilian Sea Anemone Anthopleura cascaia Using Mass Spectrometry Imaging.

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Scorpion Peptides and Ion Channels: An Insightful Review of Mechanisms and Drug Development.

The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and any disturbance in their activity can result in channelopathies, which can lead to various diseases such as autoimmune, cardiovascular, immunological, neurological, and neoplastic conditions. Given the importance of ion channels, scorpion peptides represent a valuable resource for developing drugs with targeted specificity for these channels. This review provides a comprehensive overview of the structure and classification of ion channels, the action of scorpion toxins on these channels, and potential avenues for future research. Overall, this review highlights the significance of scorpion venom as a promising source for discovering novel drugs with therapeutic potential for treating channelopathies.

Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae).

Spiders have distinct predatory behaviours selected along Araneae's evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis.

In the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a cross-reactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.

Spiders' digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family.

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Unraveling and profiling Tityus bahiensis venom: Biochemical analyses of the major toxins.

Among the scorpions found in Brazil, Tityus bahiensis is one of the species that causes most of the reported human accidents. In spite of this important constatation, the venom composition description is not available in the literature. Thus, this venom remains not properly studied, segregating this particular species into an abandoned, forgotten condition. In the present study, chromatographic separation (RP-HPLC-C18) and proteomic analyses were employed to unravel the diversity, complexity, and proportional distribution of the main peptides and proteins found in the scorpion venom. Moreover, sequence analyses and the presence of new isoforms and toxins are discussed based on a database comparison with other Tityus toxins. Our results show the presence of a wide diversity of potassium and sodium channel toxins and enzymes, such as metallopeptidases and hyaluronidases, as previously described for other species. However, the current work also describes for the first time, at the protein level, phospholipase, angiotensin-converting enzyme, cysteine-rich proteins, serine peptidase inhibitors peptides, and antimicrobial peptides. Finally, thorough data analyses allowed the description of the venom toxins distribution regarding their diversity and relative quantity. SIGNIFICANCE: The work presents the first Tityus bahiensis proteome. We have focused on describing the neurotoxin variability in terms of their isoforms/amino acid substitutions. Understanding the natural variations in the toxins' sequences is essential, once the affinity of these peptides to their respective receptors/ionic channels will vary depending on the specific peptide sequences. Moreover, the current study describes some proteins present in the venom, including enzymes being described for the first time in scorpion venoms, such as PLA2 and ACE. Moreover, we describe the individual relative quantity distribution for the different protein classes identified, as well as their variability in the T.bahiensis venom. Finally, this study also reports the development of a simple straightforward chromatographic method for scorpion venom fractionation.

Proteomic analyses of venom from a Spider Hawk, Pepsis decorata

Background: The composition of the venom from solitary wasps is poorly known, although these animals are considered sources of bioactive substances. Until the present moment, there is only one proteomic characterization of the venom of wasps of the family Pompilidae and this is the first proteomic characterization for the genus Pepsis. Methods: To elucidate the components of Pepsis decorata venom, the present work sought to identify proteins using four different experimental conditions, namely: (A) crude venom; (B) reduced and alkylated venom; (C) trypsin-digested reduced and alkylated venom, and; (D) chymotrypsin-digested reduced and alkylated venom. Furthermore, three different mass spectrometers were used (Ion Trap-Time of Flight, QuadrupoleTime of Flight, and Linear Triple Quadruple). Results: Proteomics analysis revealed the existence of different enzymes related to the insect’s physiology in the venom composition. Besides toxins, angiotensin-converting enzyme (ACE), hyaluronidase, and Kunitz-type inhibitors were also identified. Conclusion: The data showed that the venom of Pepsis decorata is mostly composed of proteins involved in the metabolism of arthropods, as occurs in parasitic wasps, although some classical toxins were recorded, and among them, for the first time, ACE was found in the venom of solitary wasps. This integrative approach expanded the range of compounds identified in protein analyses, proving to be efficient in the proteomic characterization of little-known species. It is our understanding that the current work will provide a solid base for future studies dealing with other Hymenoptera venoms.

Spatial distribution and biochemical characterization of serine peptidase inhibitors in the venom of the brazilian sea anemone anthopleura cascaia using mass spectrometry imaging

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Spiders digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Scorpion peptides and ion channels: an insightful review of mechanisms and drug development

The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and any disturbance in their activity can result in channelopathies, which can lead to various diseases such as autoimmune, cardiovascular, immunological, neurological, and neoplastic conditions. Given the importance of ion channels, scorpion peptides represent a valuable resource for developing drugs with targeted specificity for these channels. This review provides a comprehensive overview of the structure and classification of ion channels, the action of scorpion toxins on these channels, and potential avenues for future research. Overall, this review highlights the significance of scorpion venom as a promising source for discovering novel drugs with therapeutic potential for treating channelopathies.

Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis

In the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a cross-reactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.

Batroxin I: a novel Bradykinin-potentiating peptide with Cytotoxic activity isolated from Bothrops atrox snake venom

Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.

Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae)

Spiders have distinct predatory behaviours selected along Araneae’s evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Cone snail species off the Brazilian coast and their venoms: a review and update

The genus Conus includes over 900 species of marine invertebrates known as cone snails, whose venoms are among the most powerful described so far. This potency is mainly due to the concerted action of hundreds of small bioactive peptides named conopeptides, which target different ion channels and membrane receptors and thus interfere with crucial physiological processes. By swiftly harpooning and injecting their prey and predators with such deadly cocktails, the slow-moving cone snails guarantee their survival in the harsh, competitive marine environment. Each cone snail species produces a unique venom, as the mature sequences of conopeptides from the venoms of different species share very little identity. This biochemical diversity, added to the numerous species and conopeptides contained in their venoms, results in an immense biotechnological and therapeutic potential, still largely unexplored. That is especially true regarding the bioprospection of the venoms of cone snail species found off the Brazilian coast - a region widely known for its biodiversity. Of the 31 species described in this region so far, only four - Conus cancellatus, Conus regius, Conus villepinii, and Conus ermineus - have had their venoms partially characterized, and, although many bioactive molecules have been identified, only a few have been actually isolated and studied. In addition to providing an overview on all the cone snail species found off the Brazilian coast to date, this review compiles the information on the structural and pharmacological features of conopeptides and other molecules identified in the venoms of the four aforementioned species, paving the way for future studies.

Unraveling and profiling Tityus bahiensis venom: biochemical analyses of the major toxins

Among the scorpions found in Brazil, Tityus bahiensis is one of the species that causes most of the reported human accidents. In spite of this important constatation, the venom composition description is not available in the literature. Thus, this venom remains not properly studied, segregating this particular species into an abandoned, forgotten condition. In the present study, chromatographic separation (RP-HPLC-C18) and proteomic analyses were employed to unravel the diversity, complexity, and proportional distribution of the main peptides and proteins found in the scorpion venom. Moreover, sequence analyses and the presence of new isoforms and toxins are discussed based on a database comparison with other Tityus toxins. Our results show the presence of a wide diversity of potassium and sodium channel toxins and enzymes, such as metallopeptidases and hyaluronidases, as previously described for other species. However, the current work also describes for the first time, at the protein level, phospholipase, angiotensin-converting enzyme, cysteine-rich proteins, serine peptidase inhibitors peptides, and antimicrobial peptides. Finally, thorough data analyses allowed the description of the venom toxins distribution regarding their diversity and relative quantity. Significance: the work presents the first Tityus bahiensis proteome. We have focused on describing the neurotoxin variability in terms of their isoforms/amino acid substitutions. Understanding the natural variations in the toxins' sequences is essential, once the affinity of these peptides to their respective receptors/ionic channels will vary depending on the specific peptide sequences. Moreover, the current study describes some proteins present in the venom, including enzymes being described for the first time in scorpion venoms, such as PLA2 and ACE. Moreover, we describe the individual relative quantity distribution for the different protein classes identified, as well as their variability in the T.bahiensis venom. Finally, this study also reports the development of a simple straightforward chromatographic method for scorpion venom fractionation.