Physiological suppression of the larval parasitoid Glyptapanteles pallipes by the polyembryonic parasitoid Copidosoma floridanum

Precocious larvae, clonally produced together with reproductive siblings in the polyembryonic parasitoid Copidosoma floridanum, are known to physically attack competitors in multiparasitized hosts. In this study, we show that physiological suppression by C. floridanum, as well as precocious larval activity, causes death of the larval parasitoid Glyptapanteles pallipes. Approximately 70% of the hosts multiparasitized by C. floridanum and G. pallipes produced C. floridanum offspring, irrespective of the interval of multiparasitism. G. pallipes eggs or larvae died even in multiparasitized hosts that did not contain precocious larvae of C. floridanum. An injection of C. floridanum-parasitized or multiparasitized-host hemolymph into G. pallipes singly-parasitized hosts paralyzed almost all G. pallipes larvae within 70 h. In vitro analysis showed that the hemolymph factor toxic to G. pallipes eggs and larvae was present in C. floridanum-parasitized hosts through their larval stages. Heating or proteinase treatment reduced its toxicity, suggesting that the factor is a protein.     

Proteomic characterization of the hyaluronidase (E.C. 3.2.1.35) from the venom of the social wasp Polybia paulista

Polybia paulista wasp venom possesses three major allergens: phospholipase A1, hyaluronidase and antigen-5. To the best of our knowledge, no hyaluronidase from the venom of Neotropical social wasps was structurally characterized up to this moment, mainly due to its reduced amount in the venom of the tropical wasp species (about 0.5% of crude venom). Four different glycoproteic forms of this enzyme were detected in the venom of the wasp Polybia paulista. In the present investigation, an innovative experimental approach was developed combining 2-D SDS-PAGE with in-gel protein digestion by different proteolytic enzymes, followed by mass spectrometry analysis under collision-induced dissociation CID) conditions for the complete assignment of the protein sequencing. Thus, the most abundant form of this enzyme in P. paulista venom, the hyaluronidase-III, was sequenced, revealing that the first 47 amino acid residues from the N-terminal region, common to other Hymenoptera venom hyaluronidases, are missing. The molecular modeling revealed that hyaluronidase-III has a single polypeptide chain, folded into a tertiary structure, presenting a central (β/α)5 core with alternation of β-strands and α-helices; the tertiary structure stabilized by a single disulfide bridge between the residues Cys189 and Cys201. The structural pattern reported for P. paulista venom hyaluronidase-III is compatible with the classification of the enzyme as member of the family 56 of glycosidase hydrolases. Moreover, its structural characterization will encourage the use of this protein as a model for future development of "component-resolved diagnosis".     

Geographical variation of mitochondrial DNA between populations of the white-clawed crayfish Austropotamobius pallipes

1. The white-clawed crayfish Austropotamobius pallipes has a widespread distribution in Europe, but since the last century its distribution has been severely affected by a combination of factors, particularly the disease crayfish plague, introduced by foreign crayfish species. At present A. pallipes is considered as vulnerable and endangered and measures are being taken in a number of countries to conserve it.
2. The application of genetics to conservation is of practical value particularly where restocking is being considered. This study assesses levels of genetic differentiation from an analysis of mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism of four populations sampled on a European scale. Six different composite haplotypes were detected among sixty-four crayfish representing the four populations. mtDNA nucleon diversity values within species ranged from 0.63% to 12.35%. A close genetic similarity was found between British and French populations of A. pallipes .
3. Results show that mtDNA can be as a suitable diagnostic marker for measuring genetic diversity between crayfish populations and that such information is of use in planning crayfish conservation strategies.     

Geographical variation of mitochondrial DNA between populations of the white-clawed crayfish Austropotamobius pallipes

1. The white-clawed crayfish Austropotamobius pallipes has a widespread distribution in Europe, but since the last century its distribution has been severely affected by a combination of factors, particularly the disease crayfish plague, introduced by foreign crayfish species. At present A. pallipes is considered as vulnerable and endangered and measures are being taken in a number of countries to conserve it.
2. The application of genetics to conservation is of practical value particularly where restocking is being considered. This study assesses levels of genetic differentiation from an analysis of mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism of four populations sampled on a European scale. Six different composite haplotypes were detected among sixty-four crayfish representing the four populations. mtDNA nucleon diversity values within species ranged from 0.63% to 12.35%. A close genetic similarity was found between British and French populations of A. pallipes .
3. Results show that mtDNA can be as a suitable diagnostic marker for measuring genetic diversity between crayfish populations and that such information is of use in planning crayfish conservation strategies.     

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista

Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH₂ (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH₂ (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the -amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.     

Assessment of natural and artificial propagation of the white-clawed crayfish (Austropotamobius pallipes species complex) in the Alpine region with nuclear and mitochondrial markers

In a joint analysis of nuclear (allozyme) and mitochondrial markers (sequence and restriction fragment length polymorphism (RFLP) analysis of the 12S and 16S genes), five genetically distinct groups of the Austropotamobius pallipes Lereboullet species complex were detected in the Alpine region. The geographical distribution of these evolutionary lineages coincided largely with several taxa (A.p. pallipes, A. berndhauseri, A.(p.) italicus) formerly defined on the basis of morphological characters. A low level of genetic variability was found within these lineages. For some populations, the combination of the two markers gave additional information about the likelihood of their natural or artificial origin. A hybrid zone was detected between A.p. pallipes and A. berndhauseri in the Lake Geneva area. The mosaic distribution of the five evolutionary lineages indicated that conservation efforts should be aimed at the level of local populations.     

Effectiveness of CID, HCD, and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods

We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides (isolated from human blood plasma) without the use of specific "enzyme rules". In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the number of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide data sets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than SEQUEST (by 1.3-2.3 fold) and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more contiguous residues (e.g., ≥ 7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide data sets that were affected by the decoy database used and mass tolerances applied (e.g., identical peptides between data sets could be limited to ~70%), while the UStags method provided the most consistent peptide data sets (>90% overlap). The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.     

Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two‐dimensional gel electrophoresis and separation by ion‐exchange and reverse‐phase high‐performance liquid chromatography followed by mass spectrometry using tanden matrix‐assisted laser desorption/ionization with time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry and electrospray ionization‐quadrupole with time‐of‐flight (ESI‐Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10‐ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

Immunological evidence for progesterone and estradiol receptors in the freshwater crayfish Austropotamobius pallipes

In this article, we present evidence for progesterone and estradiol receptors (PR and ER, respectively) in the female of the crayfish Austropotamobius pallipes. To our knowledge, this is the first report of sex steroid receptors in crustaceans. By using immunohistochemistry and Western blotting approaches and employing three different antibodies against PR (human PR, chicken PR-hinge region, and chicken PR A/B domain) and antibodies against human ER, we showed the presence of PR in the ovary and hepatopancreas and ER in the hepatopancreas of the freshwater crayfish A. pallipes. The immunological characteristics and the tissue localization suggest a relatedness with both PR and ER in vertebrates along with their involvement in the modulation of reproductive functions in this crustaceans.     

Proteome and peptidome profiling of spider venoms

Spider venoms are an important source of novel molecules with different pharmacological properties. Recent technological developments of proteomics, especially mass spectrometry, have greatly promoted the systematic analysis of spider venom. The enormous diversity of venom components between spider species and the lack of complete genome sequence, and the limited database of protein and peptide sequences make spider venom profiling a challenging task and special considerations for technical strategies are required. This review highlights recently used methods for spider venom profiling. In general, spider venom profiling can be achieved in two parts: proteome profiling of the components with molecular weights above 10 kDa, and peptidome profiling of the components with a molecular weight of 10 kDa or under through the use of different methods. Venom proteomes are rich in various enzymes, hemocyanins, toxin-like proteins and many unknown proteins. Peptidomes are dominated by peptides with a mass of 3-6 kDa with three to five disulfide bonds. Although there are some similarities in peptide superfamily types of venoms from different spider species, the venom profile of each species is unique. The linkage of the peptidomic data with that of the cDNA approach is discussed briefly. Future challenges and perspectives are also highlighted in this review.     

Cationicity-Enhanced Analogues of the Antimicrobial Peptides,AcrAP1 and AcrAP2, from the Venom of the Scorpion,Androctonus crassicauda,Display Potent Growth Modulation Effects on Human Cancer Cell Lines

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.     

Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K(+)-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2,457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be alpha-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7,522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na(+)-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.     

Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics

Protein phosphorylation modulates a myriad of biological functions, and its regulation is vital for proper cellular activity. Mass spectrometry is the enabling tool for phosphopeptide analysis, where recent instrumentation advances in both speed and sensitivity in linear ion trap and orbitrap technologies may yield more comprehensive phosphoproteomic analyses in less time. Protein phosphorylation analysis by MS relies on structural information derived through controlled peptide fragmentation. Compared with traditional, ion-trap-based collision-induced dissociation (CID), a more recent type of fragmentation termed HCD (higher energy collisional dissociation) provides beam type CID tandem MS with detection of fragment ions at high resolution in the orbitrap mass analyzer. Here we compared HCD to traditional CID for large-scale phosphorylation analyses of murine brain under three separate experimental conditions. These included a same-precursor analysis where CID and HCD scans were performed back-to-back, separate analyses of a phosphotyrosine peptide immunoprecipitation experiment, and separate whole phosphoproteome analyses. HCD generally provided higher search engine scores with more peptides identified, thus out-performing CID for back-to-back experiments for most metrics tested. However, for phosphotyrosine IPs and in a full phosphoproteome study of mouse brain, the greater acquisition speed of CID-only analyses provided larger data sets. We reconciled our results with those in direct contradiction from Nagaraj N, D'Souza RCJ et al. (J. Proteome Res. 9:6786, 2010). We conclude, for large-scale phosphoproteomics, CID fragmentation with rapid detection in the ion trap still produced substantially richer data sets, but the back-to-back experiments demonstrated the promise of HCD and orbitrap detection for the future.