RESUMO
Snake venoms are complex mixtures of different substances, proteins being their predominant components. To study the composition of venoms, methods based on chromatographic separation and mass spectrometric analysis are currently used, requiring the application of a number of sophisticated instruments. To assess the composition of snake venoms, we propose an alternative method based on Raman spectroscopy, which is an express method to study the structural features of different substances, including proteins. The method does not require preliminary preparation of the samples, which are used in small quantities; this makes Raman spectroscopy extremely attractive for venom research. In this work, we have carried out Raman spectroscopic studies on a number of dry venoms from various venomous snakes. Based on the obtained Raman spectra, with the help of mathematical methods of dimensionality reduction and clustering, differentiation of venoms reflecting their composition and the assignment of the venom producing snake to the corresponding family or even genus were performed. The proposed method can be used to analyze both the composition of and variations in venoms of different snake species, including rare and endangered ones.
Assuntos
Venenos de Serpentes , Análise Espectral Raman , Venenos de Serpentes/análise , Espectrometria de MassasRESUMO
Snake venom is a complex mixture secreted from the glands of poisonous snakesï¼ which contains proteinsï¼ peptidesï¼ lipidsï¼ nucleosidesï¼ sugarsï¼ amino acidsï¼ aminesï¼ metal ionsï¼ and other components. According to the toxicological classificationï¼ snake venoms can be classified as neurotoxinsï¼ anticoagulants and procoagulant toxinsï¼ cardiac toxinsï¼ other toxin proteinsï¼ and enzymes. Proteins and peptides are the key components of snake venom. The establishment of rapidï¼ accurate analysis and identification methods for proteins in snake venom is a prerequisite for snake venom-related forensic identificationï¼ intoxication eventsï¼ and pharmaceutical development. Until nowï¼ the classical analysis and identification methods have mainly been biochemical or immunoassays for DNA or proteinsï¼ such as polymerase chain reactionï¼ agglutination testï¼ enzyme-linked immunosorbent assayï¼ fluorescent immunoassayï¼ and various biosensing approaches. These methods have some limitations such as a high false-positive ratioï¼ low sensitivityï¼ poor anti-interference abilityï¼ and limited species discrimination capability. In recent yearsï¼ with the rapid development of mass spectrometry ï¼MSï¼ techniquesï¼ the proteomics of snake venom has also attracted much attention and has contributed to the identification of snake speciesï¼ in which non-targeted and targeted proteomics represent two main divisions. Howeverï¼ species identification via proteomics is in its infancy in forensic science. Firstï¼ the tandem MS spectra of peptide sequences are highly complexï¼ which poses a great challenge for the strict and accurate matching of peptides based on the rational speculation of MS fragmentation rules and theoretical calculations in non-targeted proteomics. Secondï¼ for the confirmation and identification of unknown substancesï¼ reference substances are commonly neededï¼ but those for snake venom are lacking. Proteomics in snake venom identification is still in progress to improve the identification confidence and clarify the identification rules. In this workï¼ a method based on nano-ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry ï¼Nano LC-MS/HRMSï¼ and size exclusion chromatography ï¼SECï¼ was developed for identifying proteins and their source speciesï¼ with strict rules for five suspected snake venom samples and their contamination in one case. Three SEC elution peaks were obtained from each of the five samplesï¼ which were lyophilized and treated with trypsin in solutionï¼ and then separated and analyzed by Nano LC-MS/HRMS. Firstï¼ the Full MS/dd MS2 mode was used for the non-targeted acquisition of peptide information in the samplesï¼ and after submission to the Swiss-Prot databaseï¼ the protein databases of Serpentesï¼ Colubroideaï¼ Elapidaeï¼ Elapinaeï¼ and Naja were contracted stepwise and compared. A total of 32 proteins from Naja atra were identified under the conditions of both peptide spectrum match and false discovery rate less than 1%ï¼ and number of characteristic peptides greater than or equal to two. All of these were derived from ten families of Naja atraï¼ mainly three-finger toxinsï¼ metalloproteinasesï¼ and phospholipase A2. Proteins D3TTC2ï¼ D5LMJ3ï¼ Q7T1K6ï¼ Q9DEQ3ï¼ and Q9YGI4 were the most common among the five samples. Finallyï¼ the parallel reaction monitoring mode was adopted to select two unique peptides for each protein for targeted verification. It was considered that a protein in the samples was truly identified when it met the strict standard "the Δm/z of at least 75% y+ and b+ ions of each unique peptide was less than 5 ppm". After these consequently proceduresï¼ we identified that all five samples contained the venom of the Naja atra. Our identification method is a systematic and strict example that can provide effective technical support for the forensic identification of snake venom poisoningï¼ as well as for pharmaceutical development toward snake venoms.
Assuntos
Proteínas , Venenos de Serpentes , Cromatografia Líquida de Alta Pressão , Venenos de Serpentes/análise , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Peptídeos , Espectrometria de Massas em Tandem , Proteoma/análiseRESUMO
Abstract Snakebite is one of the major health issues posing considerable morbidity and mortality. According to an estimate of World Health Organization (WHO) (World health organization, 2021) approximately 5 million people are bitten by several species of snakes resulting in up to 2.5 million envenomation cases annually. The mainstay of treatment for envenomation is intravenous administration of anti-snake venom. Although antivenom neutralizes the systemic effects but it does not relieve the symptoms such as venom-induced hemorrhage, necrosis and nephrotoxicity. Moreover, the use of antivenoms is associated with hypersensitivity reactions including urticaria, anaphylaxis, or serum sickness due to their heterologous property. Furthermore, stringent storage conditions and narrow specificity of antivenoms limit their use in both developed as well as developing countries. In this context, researchers have been searching for natural products and plant extracts to explore their antivenom activity along with anti-myotoxic, anti-hemorrhagic and anti-inflammatory properties. Plant remedies may prove to be an effective alternate for antivenom sera with less adverse events and better tolerability. To the best of our knowledge, this is the first comprehensive review of medicinal plants possessing anti-snake venom activities against certain species of snakes. The current review highlights the investigated plants with their phytochemical analysis to integrate the available information for future research and development of antivenom sera.
Assuntos
Plantas Medicinais/efeitos adversos , Venenos de Serpentes/análise , Antivenenos/análise , Venenos Elapídicos/isolamento & purificação , Compostos Fitoquímicos/agonistas , Mordeduras de Serpentes/classificação , Organização Mundial da Saúde , Extratos Vegetais , Administração Intravenosa/instrumentaçãoRESUMO
The Middle East and Northern Africa, collectively known as the MENA region, are inhabited by a plethora of venomous animals that cause up to 420,000 bites and stings each year. To understand the resultant health burden and the key variables affecting it, this review describes the epidemiology of snake, scorpion, and spider envenomings primarily based on heterogenous hospital data in the MENA region and the pathologies associated with their venoms. In addition, we discuss the venom composition and the key medically relevant toxins of these venomous animals, and, finally, the antivenoms that are currently in use to counteract them. Unlike Asia and sub-Saharan Africa, scorpion stings are significantly more common (approximately 350,000 cases/year) than snakebites (approximately 70,000 cases/year) and present the most significant contributor to the overall health burden of envenomings, with spider bites being negligible. However, this review also indicates that there is a substantial lack of high-quality envenoming data available for the MENA region, rendering many of these estimates speculative. Our understanding of the venoms and the toxins they contain is also incomplete, but already presents clear trends. For instance, the majority of snake venoms contain snake venom metalloproteinases, while sodium channel-binding toxins and potassium channel-binding toxins are the scorpion toxins that cause most health-related challenges. There also currently exist a plethora of antivenoms, yet only few are clinically validated, and their high cost and limited availability present a substantial health challenge. Yet, some of the insights presented in this review might help direct future research and policy efforts toward the appropriate prioritization of efforts and aid the development of future therapeutic solutions, such as next-generation antivenoms.
Assuntos
Picadas de Escorpião/fisiopatologia , Venenos de Escorpião/análise , Mordeduras de Serpentes/fisiopatologia , Venenos de Serpentes/análise , África do Norte , Animais , Antivenenos/uso terapêutico , Humanos , Oriente Médio , Picadas de Escorpião/tratamento farmacológico , Escorpiões , Mordeduras de Serpentes/tratamento farmacológico , Mordeduras de Serpentes/terapia , SerpentesRESUMO
Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.
Assuntos
Colorimetria/métodos , Citotoxinas/análise , Elapidae/imunologia , Imunoensaio/métodos , Imunotoxinas/análise , Venenos de Serpentes/análise , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Bungarus/genética , Bungarus/fisiologia , Citotoxinas/genética , Citotoxinas/imunologia , Venenos Elapídicos/análise , Venenos Elapídicos/genética , Venenos Elapídicos/imunologia , Elapidae/fisiologia , Imunotoxinas/genética , Imunotoxinas/imunologia , Naja naja/imunologia , Naja naja/fisiologia , Venenos de Serpentes/imunologia , Viperidae/imunologia , Viperidae/fisiologiaRESUMO
The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.
Assuntos
Boidae , Cobras Corais , Fosfolipases A2/toxicidade , Proteínas de Répteis/toxicidade , Venenos de Serpentes/química , Simpatria , Peçonhas/química , Animais , Fosfolipases A2/química , Comportamento Predatório , Proteínas de Répteis/química , Venenos de Serpentes/análise , Venenos de Serpentes/enzimologia , Peçonhas/análise , Peçonhas/enzimologiaRESUMO
This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.
Assuntos
Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Venenos de Serpentes/análise , Viperidae/metabolismo , Animais , Evolução Biológica , Perfilação da Expressão Gênica/métodos , Filogeografia , Proteoma/genética , Venenos de Serpentes/química , Especificidade da Espécie , Viperidae/classificação , Viperidae/genéticaRESUMO
Deinagkistrodon acutus (D. acutus), also known as the Chinese moccasin, is a viper species found throughout the southeastern parts of China, northern Vietnam and Laos. D. acutus envenomation can result in coagulopathy and lead to death if not treated correctly. A 20-year-old man was discovered with a severely swollen left thigh with overlying dark purple, discolored skin. He was immediately transported to hospital. Laboratory examinations revealed dysfunctional coagulation and fluid-electrolyte imbalances. He died 2 h later despite resuscitation efforts. Surveillance footage revealed that he had walked through a grass field while returning home that night. Autopsy and pathological examination findings revealed a large area of muscle necrosis of the left thigh, renal tubular necrosis, and hepatocyte necrosis. Potential fang marks were found on the decedent's jeans. Due to our suspicions, we performed specific enzyme-linked immunosorbent assays (ELISA) and detected D. acutus venom in the kidneys, left thigh muscle, liver, lung, spleen, and heart tissues of the decedent. In conclusion, the clinical manifestations, autopsy, histopathological examination, ELISA, and investigation results confirmed D. acutus envenomation.
Assuntos
Agkistrodon , Edema/etiologia , Extremidade Inferior/patologia , Mordeduras de Serpentes/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática , Evolução Fatal , Humanos , Rim/química , Fígado/química , Pulmão/química , Masculino , Músculo Esquelético/química , Músculo Esquelético/patologia , Miocárdio/química , Necrose , Venenos de Serpentes/análise , Baço/química , Adulto JovemRESUMO
Identification of snake venoms is a vital step in the treatment of fatal snakebites. In this study, we use the gold-thiolate interaction between a cysteine residue and gold nanoparticles to establish a SERS method for the differentiation of the venoms of Trimeresurus stejnegeri and Bungarus multicinctus. We confirm the preference of gold nanoparticles over silver for the SERS study of snake venoms by a binding experiment that also functions to differentiate the two venom samples by colorimetry and UV-vis spectroscopy. We report the SERS spectra of Trimeresurus stejnegeri and Bungarus multicinctus venoms for the first time. The spectra display distinct SERS signatures of the snake venoms on bone-shaped gold nanoparticles made with a house recipe. These signatures correlate to selected segments of the venom proteins due to the anchoring effect of the gold-cysteine bond. The method is quick as it accomplishes in situ isolation of the structure of interest to avoid tedious purification of the samples. The location of the interactive cysteine residue makes a novel characteristic of proteins in general.
Assuntos
Cisteína/análise , Ouro/análise , Nanopartículas Metálicas/análise , Venenos de Serpentes/análise , Análise Espectral Raman/métodos , Animais , Bungarus , Colorimetria/métodos , Venenos de Crotalídeos , Cisteína/química , Ouro/química , Nanopartículas Metálicas/química , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificaçãoRESUMO
BACKGROUND: Detection and quantification of snake venom in envenomed patients' blood is important for identifying the species responsible for the bite, determining administration of antivenom, confirming whether sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Currently, snake venom detection is not available in clinical practice in Sri Lanka. This study describes the development of enzyme immunoassays (EIA) to differentiate and quantify venoms of Russell's viper (Daboia russelii), saw-scaled viper (Echis carinatus), common cobra (Naja naja), Indian krait (Bungarus caeruleus), and hump-nosed pit viper (Hypnale hypnale) in the blood of envenomed patients in Sri Lanka. METHODOLOGY / PRINCIPAL FINDINGS: A double sandwich EIA of high analytical sensitivity was developed using biotin-streptavidin amplification for detection of venom antigens. Detection and quantification of D. russelii, N. naja, B. caeruleus, and H. hypnale venoms in samples from envenomed patients was achieved with the assay. Minimum (less than 5%) cross reactivity was observed between species, except in the case of closely related species of the same genus (i.e., Hypnale). Persistence/ recurrence of venom detection following D. russelii envenoming is also reported, as well as detection of venom in samples collected after antivenom administration. The lack of specific antivenom for Hypnale sp envenoming allowed the detection of venom antigen in circulation up to 24 hours post bite. CONCLUSION: The EIA developed provides a highly sensitive assay to detect and quantify five types of Sri Lankan snake venoms, and should be useful for toxinological research, clinical studies, and forensic diagnosis.
Assuntos
Técnicas Imunoenzimáticas , Mordeduras de Serpentes/sangue , Mordeduras de Serpentes/diagnóstico , Venenos de Serpentes/análise , Adolescente , Adulto , Animais , Antivenenos/uso terapêutico , Reações Cruzadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mordeduras de Serpentes/tratamento farmacológico , Serpentes , Sri Lanka , Adulto JovemRESUMO
Snake venoms are inherently complex. They are mixtures of multiple enzymes, peptides, lipids, carbohydrates, nucleosides, and metal ions. Metal ions make up a small portion of a snake's venom but play outsized roles in enzyme function and stability. Unlike enzyme primary structure, which is easily predicted from genomic sequences, a venom's metal ion content must be measured directly. We leveraged the high throughput and sensitivity of inductively coupled plasma mass spectrometry to analyze the metal ion content of seven North American snake venoms. All venoms were collected from snakes reared at one location, so we could discount variation from environmental or geographical factors. We profiled 71 metal isotopes. Selenium isotopes were consistently high across all venoms tested. When each venom's toxicity was graphed as a function of each different metal isotope, the only strong relationships between metal content and toxicity were for magnesium isotopes.
Assuntos
Venenos de Serpentes/análise , Animais , Espectrometria de Massas , Peptídeos , Proteômica , Serpentes , Estados UnidosRESUMO
Next-generation sequencing (NGS), particularly RNA-sequencing (RNA-Seq) technique, allows detection and quantification of different RNA transcripts in a tissue sample, and in our case toxin transcripts from snake venom glands. Using this approach, novel toxin transcripts can be detected and abundancies of different isoforms of each toxin measured. The analytical pipeline can be briefly outlined as follows. Isolation of mRNA from tissue under RNase-free condition is essential to keep mRNA intact before sequencing. After mRNA fragmentation, the adapters are added to both ends of the fragments to synthesize complementary cDNAs. The obtained cDNA library is then sequenced on Illumina HiSeq 2000 platform. Quality of millions of reads produced from the NGS is checked and the sequences corresponding to the adapters and low-quality reads are removed. Subsequently, the NGS data are subjected to the workflow of de novo assembly, quantification of expression levels, annotation of transcripts, and identification of ORFs, signal peptides, structurally conserved domains, and functional motifs. In this report we describe the listed methodological steps and techniques in details and refer to the platforms and software that may be adopted for similar studies.
Assuntos
Glândulas Exócrinas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Venenos de Serpentes/análise , Animais , Biblioteca GênicaRESUMO
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.
Assuntos
Cristalografia por Raios X/métodos , Venenos de Serpentes/análise , Venenos de Aranha/análise , Animais , Peptídeos/análise , Proteínas/análiseRESUMO
Integrin targeting has been shown to be an effective approach for anticancer therapy. We engineered a recombinant disintegrin, vicrostatin (VCN), that binds with high affinity and specificity to the Arg-Gly-Asp (RGD) class of integrins, including αvß3, αvß5, and α5ß1, involved in tumor invasion and metastasis. We used three different delivery modalities to examine anticancer activity of VCN in mouse models of human ovarian cancer, glioma, and prostate cancer. A female mouse model was used to examine the treatment of established ovarian cancer (OC) using VCN delivered intraperitoneally (IP) weekly either in saline or impregnated in a viscoelastic gel. SKOV3luc cells (a human OC cell line) were directly injected IP into immunodeficient mice. We also examined the antitumor activity of radioiodinated VCN delivered intravenously in a human glioma model in nude mice. We evaluated the effectiveness of 131I-VCN in combination with the DNA alkylating agent temozolomide in limiting glioma growth. Finally, treatment of a bone metastatic model of human prostate cancer (PC) in immunodeficient mice was examined using a liposomal formulation of VCN (LVCN) delivered intravenously. Human PC cells were suspended in a solution of Matrigel and injected into the left tibia of immunodeficient mice. Diameters of both the left and right (control) tibias were measured by caliper repeatedly after VCN treatment was initiated.
Assuntos
Desintegrinas/análise , Glioma/metabolismo , Neoplasias Ovarianas/metabolismo , Venenos de Serpentes/análise , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos NusRESUMO
Snake venoms could lead to the development of new drugs to treat a range of life-threatening conditions like cardiovascular diseases. Most snake venoms contain a large variety of lethal toxins as well as anti-adhesive proteins such as disintegrins, which have evolved from the harmless compounds ADAMs (proteins with a disintegrin and a metalloprotease domain) and C-type lectin proteins which disturb connective tissue and cell-matrix interaction. These anti-adhesive proteins target and block integrin receptors and disrupt normal biological processes in snakes' prey such as connective tissue physiology and blood clotting. This chapter provides the experimental details of a practical, cell-based adhesion protocol to help identify and isolate disintegrins and C-type lectin proteins from snake venoms, important tools in integrin research and lead compounds for drug discovery.
Assuntos
Adesão Celular/fisiologia , Venenos de Serpentes/análise , Animais , Bioensaio , Coagulação Sanguínea/efeitos dos fármacos , Desintegrinas/antagonistas & inibidores , Humanos , Lectinas/química , Lectinas/farmacologiaRESUMO
Animal venoms are used as defense mechanisms or to immobilize and digest prey. In fact, venoms are complex mixtures of enzymatic and non-enzymatic components with specific pathophysiological functions. Peptide toxins isolated from animal venoms target mainly ion channels, membrane receptors and components of the hemostatic system with high selectivity and affinity. The present review shows an up-to-date survey on the pharmacology of snake-venom bioactive components and evaluates their therapeutic perspectives against a wide range of pathophysiological conditions. Snake venoms have also been used as medical tools for thousands of years especially in tradition Chinese medicine. Consequently, snake venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. For instance, Captopril® (Enalapril), Integrilin® (Eptifibatide) and Aggrastat® (Tirofiban) are drugs based on snake venoms, which have been approved by the FDA. In addition to these approved drugs, many other snake venom components are now involved in preclinical or clinical trials for a variety of therapeutic applications. These examples show that snake venoms can be a valuable source of new principle components in drug discovery.
Assuntos
Descoberta de Drogas , Venenos de Serpentes/análise , Animais , Anticoagulantes/farmacologia , Ensaios Clínicos como Assunto , Desenvolvimento de Medicamentos , Neurotoxinas/farmacologia , Filogenia , Venenos de Serpentes/farmacologia , Serpentes/classificaçãoRESUMO
Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).
Assuntos
Proteoma , Proteômica , Venenos de Serpentes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Eletroforese em Gel de Poliacrilamida , Peptídeos/análise , Peptídeos/metabolismo , Proteômica/métodos , Venenos de Serpentes/análise , Espectrometria de Massas em TandemRESUMO
The venom produced by snakes contains complex mixtures of pharmacologically active proteins and peptides which play a crucial role in the pathophysiology of snakebite diseases. The deep understanding of venom proteomes can help to improve the treatment of this "neglected tropical disease" (as expressed by the World Health Organization [WHO]) and to develop new drugs. The most widely used technique for venom analysis is liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bottom-up (BU) proteomics. Considering the fact that multiple multi-locus gene families encode snake venom proteins, the major challenge for the BU proteomics is the limited sequence coverage and also the "protein inference problem" which result in a loss of information for the identification and characterization of toxin proteoforms (genetic variation, alternative mRNA splicing, single nucleotide polymorphism [SNP] and post-translational modifications [PTMs]). In contrast, intact protein measurements with top-down (TD) MS strategies cover almost complete protein sequences, and prove the ability to identify venom proteoforms and to localize their modifications and sequence variations.
Assuntos
Proteoma , Proteômica , Venenos de Serpentes , Animais , Cromatografia Líquida de Alta Pressão , Proteoma/análise , Proteoma/química , Análise de Sequência de Proteína , Venenos de Serpentes/análise , Venenos de Serpentes/química , Espectrometria de Massas em TandemRESUMO
This chapter briefly discusses the developments in electrophoresis of proteins from Tiselius' moving-boundary electrophoresis to the modern-day two-dimensional polyacrylamide gel electrophoresis. It also touches upon the staining methods used to visualize total proteins post electrophoresis.
Assuntos
Eletroforese/métodos , Venenos de Serpentes/análise , Serpentes/metabolismo , Animais , Eletroforese Capilar , Eletroforese em Gel Bidimensional , Focalização IsoelétricaRESUMO
High-throughput technologies were used to identify venom gland toxin expression and to characterize the venom proteomes of two rear-fanged snakes, Ahaetulla prasina (Asian Green Vine Snake) and Borikenophis portoricensis (Puerto Rican Racer). Sixty-nine complete toxin-coding transcripts from 12 venom protein superfamilies (A. prasina) and 50 complete coding transcripts from 11 venom protein superfamilies (B. portoricensis) were identified in the venom glands. However, only 18% (A. prasina) and 32% (B. portoricensis) of the translated protein isoforms were detected in the proteome of these venoms. Both venom gland transcriptomes and venom proteomes were dominated by P-III metalloproteinases. Three-finger toxins, cysteine-rich secretory proteins, and C-type lectins were present in moderate amounts, but other protein superfamilies showed very low abundances. Venoms contained metalloproteinase activity comparable to viperid snake venom levels, but other common venom enzymes were absent or present at negligible levels. Western blot analysis showed metalloproteinase and cysteine-rich secretory protein epitopes shared with the highly venomous Boomslang (Dispholidus typus). The abundance of metalloproteinases emphasizes the important trophic role of these toxins. Comprehensive, transcriptome-informed definition of proteomes and functional characterization of venom proteins in rear-fanged snake families help to elucidate toxin evolution and provide models for protein structure-function analyses.