Toxinology in the proteomics era: a review on arachnid venom proteomics

Abstract The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000s. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms (scorpion venom) AND (proteome) for scorpion venomics, and (spider venom) AND (proteome) for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.

Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

Overview of protein posttranslational modifications in Arthropoda venoms

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.(AU)

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah

King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.(AU)

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah

Abstract King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Abstract Background Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.

Proteomics, toxicity and antivenom neutralization of Sri Lankan and Indian Russell's viper (Daboia russelii) venoms

The western Russell's viper (Daboia russelii) is widely distributed in South Asia, and geographical venom variation is anticipated among distant populations. Antivenoms used for Russell's viper envenomation are, however, raised typically against snakes from Southern India. The present study investigated and compared the venom proteomes of D. russelii from Sri Lanka (DrSL) and India (DrI), the immunorecognition of Indian VINS Polyvalent Antivenom (VPAV) and its efficacy in neutralizing the venom toxicity. Methods: The venoms of DrSL and DrI were decomplexed with C18 high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins fractionated were identified through nano-ESI-liquid chromatography-tandem mass spectrometry (LCMS/MS). The immunological studies were conducted with enzyme-linked immunosorbent assay. The neutralization of the venom procoagulant effect was evaluated in citrated human plasma. The neutralization of the venom lethality was assessed in vivo in mice adopting the WHO protocol. Results: DrSL and DrI venom proteomes showed comparable major protein families, with phospholipases A2 (PLA2) being the most abundant (> 60% of total venom proteins) and diverse (six protein forms identified). Both venoms were highly procoagulant and lethal (intravenous median lethal dose in mice, LD50 = 0.24 and 0.32 µg/g, for DrSL and DrI, respectively), while lacking hemorrhagic and anticoagulant activities. VPAV was immunoreactive toward DrSL and DrI venoms, indicating conserved protein antigenicity in the venoms. The high molecular weight venom proteins were, however, more effectively immunorecognized than small ones. VPAV was able to neutralize the coagulopathic and lethal effects of the venoms moderately. Conclusion: Considering that a large amount of venom can be injected by Russell's viper during envenomation, the potency of antivenom can be further improved for optimal neutralization and effective treatment. Region-specific venoms and key toxins may be incorporated into the immunization procedure during antivenom production.(AU)

Protein structure of the venom in nine species of snake: from bio-compounds to possible healing agents

Due to its various structures in bio-compounds, snake venom is the indisputable result of evolutionary stages of molecules with an increasingly complex structure, high specificity, and of great importance for medicine because of their potential. The present study proposed an underpinning examination of venom composition from nine species of venomous snakes using a useful and replicable methodology. The objective was the extension of the evaluation of protein fractions in the field up to 230 kDa to permit possible identification of some fractions that are insufficiently studied. The gel capillary electrophoresis method on the chip was performed using an Agilent 2100 bioassay with the 80 and 230-LabChip Protein kits. Interpretation of electrophoresis was performed using the Protein 2100 expert (Agilent) test software as follows: a) Protein 80 (peak size scale): 1.60, 3.5, 6.50, 15.00, 28.00, 46.00, 63.00, 95.00 kDa; b) Protein 230 (peak size scale): 4.50, 7.00, 15.00, 28.00, 46.00, 63.00, 95.00, 150.00, 240.00 kDa. The screening revealed the presence of compounds with a molecular weight greater than 80 kDa, in the case of Vipera aspis and Vipera xantina palestinae. For V. aspis, a 125 kDa molecular weight pro-coagulant protein was identified, known as being involved in the reduction of plasma clotting time without any direct activity in the fibrinogen coagulation process. The samples examined on the Protein 230-LabChip electrophoresis chip can be considered as a novelty with possible uses in medicine, requiring further approaches by advanced proteomics techniques to confirm the intimate structural features and biological properties of snake venoms.

Proteomics and antivenom immunoprofiling of Russell's viper (Daboia siamensis) venoms from Thailand and Indonesia

The Eastern Russell's viper, Daboia siamensis, is a WHO Category 1 medically important venomous snake. It has a wide but disjunct distribution in Southeast Asia. The specific antivenom, D. siamensis Monovalent Antivenom (DsMAV-Thailand) is produced in Thailand but not available in Indonesia, where a heterologous trivalent antivenom, Serum Anti Bisa Ular (SABU), is used instead. This study aimed to investigate the geographical venom variation of D. siamensis from Thailand (Ds-Thailand) and Indonesia (Ds-Indonesia), and the immunorecognition of the venom proteins by antivenoms. Methods: The venom proteins were decomplexed with reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by in-solution tryptic digestion, nano-liquid chromatography-tandem mass spectrometry and protein identification. The efficacies of DsMAV-Thailand and SABU in binding the various venom fractions were assessed using an enzyme-linked immunosorbent assay optimized for immunorecognition profiling. Results: The two most abundant protein families in Ds-Thailand venom are phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitor (KSPI). Those abundant in Ds-Indonesia venom are PLA2 and serine protease. KSPI and vascular endothelial growth factor were detected in Ds-Thailand venom, whereas L-amino acid oxidase and disintegrin were present in Ds-Indonesia venom. Common proteins shared between the two included snaclecs, serine proteases, metalloproteinases, phosphodiesterases, 5'nucleotidases and nerve growth factors at varying abundances. DsMAV-Thailand exhibited strong immunorecognition of the major protein fractions in both venoms, but low immunoreactivity toward the low molecular weight proteins e.g. KSPI and disintegrins. On the other hand, SABU was virtually ineffective in binding all fractionated venom proteins. Conclusion: D. siamensis venoms from Thailand and Indonesia varied geographically in the protein subtypes and abundances. The venoms, nevertheless, shared conserved antigenicity that allowed effective immunorecognition by DsMAV-Thailand but not by SABU, consistent with the neutralization efficacy of the antivenoms. A specific, appropriate antivenom is needed in Indonesia to treat Russell's viper envenomation.(AU)

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. Conclusion: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.(AU)

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)

Protein-species quantitative venomics: looking through a crystal ball

In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term "protein species" is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics "Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts" published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors' view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.(AU)

Strategies in 'snake venomics' aiming at an integrative view of compositional, functional, and immunological characteristics of venoms

This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.(AU)

It is time for top-down venomics

The protein composition of animal venoms is usually determined by peptide-centric proteomics approaches (bottom-up proteomics). However, this technique cannot, in most cases, distinguish among toxin proteoforms, herein called toxiforms, because of the protein inference problem. Top-down proteomics (TDP) analyzes intact proteins without digestion and provides high quality data to identify and characterize toxiforms. Denaturing top-down proteomics is the most disseminated subarea of TDP, which performs qualitative and quantitative analyzes of proteoforms up to ~30 kDa in high-throughput and automated fashion. On the other hand, native top-down proteomics provides access to information on large proteins (> 50 kDA) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry. The use of native and denaturing top-down venomics introduced novel and useful techniques to toxinology, allowing an unprecedented characterization of venom proteins and protein complexes at the toxiform level. The collected data contribute to a deep understanding of venom natural history, open new possibilities to study the toxin evolution, and help in the development of better biotherapeutics.(AU)

Protein-species quantitative venomics: looking through a crystal ball

Abstract In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term protein species is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.

Strategies in snake venomics aiming at an integrative view of compositional, functional, and immunological characteristics of venoms

Abstract This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.

It is time for top-down venomics

Abstract The protein composition of animal venoms is usually determined by peptide-centric proteomics approaches (bottom-up proteomics). However, this technique cannot, in most cases, distinguish among toxin proteoforms, herein called toxiforms, because of the protein inference problem. Top-down proteomics (TDP) analyzes intact proteins without digestion and provides high quality data to identify and characterize toxiforms. Denaturing top-down proteomics is the most disseminated subarea of TDP, which performs qualitative and quantitative analyzes of proteoforms up to ~30 kDa in high-throughput and automated fashion. On the other hand, native top-down proteomics provides access to information on large proteins (> 50 kDA) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry. The use of native and denaturing top-down venomics introduced novel and useful techniques to toxinology, allowing an unprecedented characterization of venom proteins and protein complexes at the toxiform level. The collected data contribute to a deep understanding of venom natural history, open new possibilities to study the toxin evolution, and help in the development of better biotherapeutics.

Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity

Background Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs.Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims.Methods In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species.Results Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria.Conclusions This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides fromL. abdullahbayrami venom.(AU)