Zhigui Duan

Proteomic analysis of the venom from the scorpion Mesobuthus martensii.

UNLABELLED The scorpion Mesobuthus martensii is the most populous species in eastern Asian countries, and several toxic components have been identified from their venoms. Nevertheless, a complete proteomic profile of the venom of M. martensii is still not available. In this study, the venom of M. martensii was analyzed by comprehensive proteomic approaches. 153 fractions were isolated from the M. martensii venom by 2-DE, SDS-PAGE and RP-HPLC. The ESI-Q-TOF MS results of all fractions were used to search the scorpion genomic and transcriptomic databases. Totally, 227 non-redundant protein sequences were unambiguously identified, composed of 134 previously known and 93 previously unknown proteins. Among 134 previously known proteins, 115 proteins were firstly confirmed from the M. martensii crude venom and 19 toxins were confirmed once again, involving 43 typical toxins, 7 atypical toxins, 12 venom enzymes and 72 cell associated proteins. In typical toxins, 7 novel-toxin sequences were identified, including 3 Na(+)-channel toxins, 3K(+)-channel toxins and 1 no-annotation toxin. These results increased 230% (115/50) venom components compared with previous studies from the M. martensii venom, especially 50% (24/48) typical toxins. Additionally, a mass fingerprint obtained by MALDI-TOF MS indicated that the scorpion venom contained more than 200 different molecular mass components. BIOLOGICAL SIGNIFICANCE This work firstly gave a systematic investigation of the M. martensii venom by combined proteomics strategy coupled with genomics and transcriptomics. A large number of protein components were unambiguously identified from the venom of M. martensii, most of which were confirmed for the first time. We also contributed 7 novel-toxin sequences and 93 protein sequences previously unknown to be part of the venom, for which we assigned potential biological functions. Besides, we obtained a mass fingerprint of the M. martensii venom. Together, our study not only provides the most comprehensive catalog of the molecular diversity of the M. martensii venom at the proteomic level, but also enriches the composition information of scorpion venom.

A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing.

Physiological and biochemical analysis of L. tredecimguttatus venom collected by electrical stimulation.

The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 μg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.ResumenEn el presenta trabajo, se analiza el veneno de Latrodectus tredecimguttatus obtenido por estimulación eléctrica, método de recolección que evita la contaminación. Tras aplicación de técnicas de HPLC, espectrometría de masas y electroforesis en gel se deduce que el veneno contiene predominantemente proteínas ácidas de peso molecular superior a 10kDa. Las mas abundantes se concentran alrededor de 100 kDa, entre las que se encuentra la latrocina, principal componente tóxico del veneno. El análisis enzimático del veneno indica que incluye actividad enzimática de varios tipos de hidrolasas, incluyendo hialuronidasa y proteasas. Tras inyección del veneno en el ratón y en la cucaracha Periplaneta americana se obtuvieron valores de LD50 de 0,16 mg/kg y 1,87 microg/g respectivamente. Los experimentos electrofisiológicos mostraron que el veneno bloqueaba la transmisión neuromuscular tanto en preparado nervio frénico-hemidiafragma de ratón como en el de vas deferens de rata. La fracción del veneno que contenía péptidos de bajo peso molecular (inferior a 10 kDa) era inefectiva sobre la transmisión. En suma, los resultados muestran que la gran toxicidad del veneno de Latrodectus tredecimguttatus se relaciona con su contenido en proteínas de elevado peso molecular, cuyas actividades hidrolíticas están incluidas en el lactrodectismo.

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

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

Protein compositional analysis of the eggs of black widow spider (Latrodectus tredecimguttatus): implications for the understanding of egg toxicity.

Previous work found that high‐molecular‐weight fractions in the egg extract of Latrodectus tredecimguttatus exhibited strong toxicities. For investigating the possible relationship of proteins in the eggs with the toxic effect, the protein composition of the eggs was analyzed using proteomic strategies and compared with that of the spiders venom. SDS‐PAGE showed that the proteins of eggs were primarily distributed in the molecular weight range of higher than 55 kDa as well as around 34 kDa, having high abundance proteins with molecular weights of about 60 kDa and 130 kDa. A total of 157 proteins were identified from the egg extract, which were involved in important cellular functions and processes including catalysis, transport, and metabolism regulation. Comparison indicated that the protein composition of eggs is more complex than that of venom, and there are few similarities between the protein composition of the two materials, demonstrating that the eggs have their own distinct toxic mechanism.

Purification and Partial Characterization of a Novel Neurotoxic Protein from Eggs of Black Widow Spiders (Latrodectus tredecimguttatus)

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion‐exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse‐isolated phrenic nerve‐hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin‐sensitive sodium current in rat dorsal root ganglion cells. The N‐terminal sequence of the protein was identified by the Edman degradation to be N‐S‐I‐A‐D‐D‐R‐Y‐R‐W‐P‐G‐Y‐P‐G‐A‐G‐L‐I‐P‐Y‐I‐I‐D‐S—. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin‐I) is a novel neurotoxic protein purified from the eggs of black widow spiders.

Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders

The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders.

Development and application of wide-range gradient gel electrophoresis to proteome analysis

SDS is widely used to treat proteins that are difficult to solubilize and digest and improve protein separation in SDS-PAGE. However, SDS interferes with subsequent analyses and needs to be removed prior to digestion and LC-MS/MS analysis, whereas the conventional SDS-PAGE lacks the ability to efficiently remove SDS and retain low-molecular-weight proteins and peptides. In the present work, we developed a wide-range gradient gel electrophoresis (WGGE) system in a vertical slab gel electrophoresis cell, which was primarily composed of a 4–20% continuous gradient polyacrylamide gel separation layer and two interception layers with even higher concentrations (30% and 50%, respectively). The main advantages of the system are simultaneously cleaning up SDS-solubilized samples, separating proteins and intercepting low-molecular-weight proteins and peptides, thereby simplifying experimental operation, improving protein recovery and enhancing the total efficiency of proteome analysis. Using this system, about 87.25% of SDS in the sample and gel was electrophoretically removed and a peptide with a molecular weight of 3.75 kDa was efficiently intercepted. Combined with CapLC-MS/MS, the WGGE system was applied to the analysis of rat liver membrane-enriched protein samples and the results indicated that the WGGE-based strategy is suitable for the identification of proteins varying in molecular weight, pI, hydrophobicity, etc., suggesting potential applications in global and comparative analyses of various proteomes.

Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

BackgroundNumerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena.ResultsHWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed.ConclusionsThis work has provided not only a new insight into the action mechanism of HWTX-IV but also a reference technology for the relevant researches.

A sample preparation method for micro-scale membrane proteome analysis

Abstract Detergents are widely used to improve the solubilization and extraction of hydrophobic membrane proteins in proteomics. Since most detergents are not compatible with subsequent steps of analysis, the removal of detergents from samples, especially those in micro-scale amounts, is a worthy topic of investigation. In this paper, we present a novel polyvinylidene difluoride (PVDF) membrane-mediated sample preparation method for micro-scale membrane proteome analysis, using a rat liver cell membrane-enriched fraction as model material. The proteins in the fraction were extracted in a 2 % sodium dodecyl sulfate (SDS) solution and the protein solution was applied on a piece of PVDF membrane followed by drying and repeated washing in order to remove SDS and other salts. Quantitative determination indicated that about 84% of the SDS in the sample was removed and protein loss was less than 10%. Four methods were used and compared for digesting the proteins adsorbed on PVDF membrane. Dimethyl formamide (DMF)-assisted digestion was the most effective with regard to the identification of membrane proteins, particularly the highly hydrophobic multi-transmembrane proteins. These results demonstrate that PVDF membrane-aided sample cleanup combined with DMF-assisted digestion has potential utility in the micro-scale membrane proteome analysis.