Proteomic and peptidomic characterization of the venom from the Chinese bird spider, Ornithoctonus huwena Wang.

The bird spider Ornithoctonus huwena Wang is a very venomous spider in China. Several compounds with different types of biological activities have been identified previously from the venom of this spider. In this study, we have performed a proteomic and peptidomic analysis of the venom. The venom was preseparated into two parts: the venom proteins with molecular weight (MW) higher than 10,000 and the venom peptides with MW lower than 10 000. Using one-dimensional gel electrophoresis (1-DE), two-dimensional gel electrophoresis (2-DE), and mass spectrometry, 90 proteins were identified, including some important enzymes, binding proteins, and some proteins with significant biological functions. For venom peptides, a combination of cation-exchange and reversed-phase chromatography was employed. More than 100 components were detected by mass spectrometry, and 47 peptides were sequenced by Edman degradation. The peptides display structural and pharmacological diversity and share little sequence similarity with peptides from other animal venoms, which indicates the venom of O. huwena Wang is unique. The venom peptides can be classified into several superfamilies. Also it is revealed that gene duplication and focal hypermutation have taken place during the evolution of the spider toxins.

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

Chinese tarantula, Chilobrachys jingzhao is one of the most venomous spiders in southern China and its venom is a mixture of various compounds with diversified biological activities. The proteome of C. jingzhao venom was analyzed by proteomic techniques. Proteins with molecular weight of over 10 kDa, indicated by gel-filtration and SDS-PAGE, were analyzed using 2-DE and MALDI-TOF/TOF and LC/ESI-Q-TOF MS. More than 90 proteins were detected, with 47 confirmed by sequence similarity search using mass spectrum driven basic local alignment search tool (MS BLAST). On the other hand, peptides with MW lower than 10 kDa were separated by HPLC and identified by MALDI-TOF MS and Edman degradation sequencing. About 120 peptides were detected, 60 of which were fully or partially sequenced. Our results indicate that peptides with MW lower than 10 kDa are the major components in the crude venom of C. jingzhao. Like those of other tarantulas, these peptides are very likely to act on various ion channels. These results pave a way for further detailed structure-function correlation analysis of the individual toxins present in the venom of C. jingzhao.

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins.

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.

Proteome analysis of combined effects of androgen and estrogen on the mouse mammary gland.

We and other investigators have previously shown that estrogen and androgen have synergistic effects on the growth of mammary epithelial ducts and alveoli in the Noble mouse. However, the molecular mechanisms behind the synergy are unknown. In the present study, we treated female FVB mice with 17-estrodial (E2) and 5-dihydrotestosterone-bezonate (DHT-B) using slow-releasing hormone pellets for 7 months. Dissection showed that hormone treatment caused atypical hyperplasia of mammary ducts and alveoli. A functional proteomic approach was used to study the holistic protein changes in mammary glands. 2-DE was used to separate proteins. Twenty-five protein spots that were differentially expressed in hormone-treated tissues compared to the control were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF-MS, which include some proteins that are correlative with response to estrogen and androgen stimulation, cells differentiation and growth, signal transduction, metabolism, etc. Real-time RT-PCR was carried out to verify the different expression. These results offered some clues to understand the function of E2 and DHT-B.

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins.

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.

DEPD: a novel database for differentially expressed proteins.

SUMMARY: The Differentially Expressed Protein Database was designed to store the output of comparative proteomics studies and provides a publicly available query and analysis platform for data mining. The database contains information about more than 3000 differentially expressed proteins (DEPs) manually extracted from the published literature, including relevant biological, experimental and methodological elements. Tools for visualization and functional analysis of DEPs are provided via a user-friendly webinterface. AVAILABILITY: http://protchem.hunnu.edu.cn/depd/.

Jingzhaotoxin-I, a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation.

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3'- and 5'-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is approximately 30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an alpha-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.

Jingzhaotoxin-III, a novel spider toxin inhibiting activation of voltage-gated sodium channel in rat cardiac myocytes.

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.

Proteome comparative analysis of gynogenetic haploid and diploid embryos of goldfish (Carassius auratus).

Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.

Function and solution structure of hainantoxin-I, a novel insect sodium channel inhibitor from the Chinese bird spider Selenocosmia hainana.

Hainantoxin-I is a novel peptide toxin, purified from the venom of the Chinese bird spider Selenocosmia hainana (=Ornithoctonus hainana). It includes 33 amino acid residues with a disulfide linkage of I-IV, II-V and III-VI, assigned by partial reduction and sequence analysis. Under two-electrode voltage-clamp conditions, hainantoxin-I can block rNa(v)1.2/beta(1) and the insect sodium channel para/tipE expressed in Xenopus laevis oocytes with IC(50) values of 68+/-6 microM and 4.3+/-0.3 microM respectively. The three-dimensional solution structure of hainantoxin-I belongs to the inhibitor cystine knot structural family determined by two-dimensional (1)H nuclear magnetic resonance techniques. Structural comparison of hainantoxin-I with those of other toxins suggests that the combination of the charged residues and a vicinal hydrophobic patch should be responsible for ligand binding. This is the first report of an insect sodium channel blocker from spider venom and it provides useful information for the structure-function relationship studies of insect sodium channels.

[Identification of protein spots in silver-stained two-dimensional gels based on chemically assisted fragmentation by MALDI-TOF-PSD mass spectrometry].

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides,accomplished with O-methylisourea hydrogen sulfate, converted lysine into homoarginine residues, improving detection in MALDI-MS. Then tryptic peptides were labeled with sulfonic acid groups at the N-termini using 3-sulfopropionic acid NHS-ester chemistry. The derivatives enhanced post-source decay (PSD) fragmentation signals and produced a spectrum containing only y-ions. This facilitated de novo peptide sequencing, so it is an important contribution to unambiguous protein identification in proteome research. This method was successfully applied in nasopharyngeal carcinoma(NPC) proteome study.

Preliminary Study on the Primary Structure of Dragline Fibroin from the Spider Araneus ventrocosus.

The amino acid composition of the dragline fibroin from the spider fibroin of the spider Araneus ventrocosus was analysed and compared with that of the fibroins from different species of spiders. By means of partial acid hydrolysis and high performance liquid chromatography, several peptide fragments of the dragline fibroin were purified. The amino acid sequence analysis showed the sequences of these peptides to be different from that of the fibroin from the spider Nephila clavipes except for one common fragment with the sequence of GYGPG.