Comparison of the medium molecular weight venom fractions from five species of common social wasps by MALDI-TOF spectra profiling

The average spectral profiles and the exact mass weight (MW) of biomolecules present in the medium fraction (from 900 to 3000 Da) of the venom of five social wasps (three European and one North American Polistes and the European hornet Vespa crabro) were determined by matrix assisted laser desorption ionization time of flight (MALDI-TOF) MS. Data were obtained analyzing the venom of single specimens (N = 46) and elaborated with the ClinProTools 2.0 (CPT) software to search for differences among the five species examined. Interesting differences in the spectral profiles were found, allowing the discrimination of venoms belonging to the different species, and their possible use as a quality control method in venom immunotherapy (VIT) for allergic patients.     

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH(2) (MW 1611.98 Da) and IDWLKLGKMVMDVL-NH(2) (MW 1658.98 Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of polymorphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging.     

Vasoactive peptides from venom of the wasp Polistes gallicus. Isolation and physicochemical and functional properties

Six vasoactive peptides have been isolated in the homogeneous form from the venom of the waspPolistes gallicus. Their physicochemical characteristics have been investigated. The wasp kinins exhibit myotropic and hypotensive effects. One of the kinins shows a prolonged hypotensive action. A. S. Sadykov Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 564–568, July–August, 1989.     

Structural analogues of bradykinin and other vasoactive peptides in the venom of the wasp Polistes gallicus

Six vasoactive peptides have been isolated in the homogeneous state from the venom of the waspPolistes gallicus. Their full amino acid sequences have been determined. It has been shown that four of them are structural analogues of bradykinin with additional N- and C-terminal sequences and intramolecular substitutions of amino acids. Some structure-function relationships in bradykinin analogues are discussed.     

Vasoactive peptides from venom of the wasp Polistes gallicus. Isolation and physicochemical and functional properties

Six vasoactive peptides have been isolated in the homogeneous form from the venom of the waspPolistes gallicus. Their physicochemical characteristics have been investigated. The wasp kinins exhibit myotropic and hypotensive effects. One of the kinins shows a prolonged hypotensive action.A. S. Sadykov Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 564–568, July–August, 1989.     

De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis

Antimicrobial peptides (AMPs), named lycocitin 1, 2 and 3, and a peptide with a monoisotopic molecular mass of 3038.70 Da were detected in the venom glands of the wolf spider Lycosa singoriensis. Two of the peptides, lycocitin 1 and 2, are new AMPs whereas lycocitin 3 is highly homologous to lycotoxin II isolated from the venom of spider Lycosa carolinensis. In addition, two other peptides with monoisotopic masses of 2034.20 and 2340.28 Da showing the motif typical for antimicrobial peptides were also identified. These peptides and lycocitin 1, 2 and 3 were de novo sequenced using electron capture dissociation and low-energy collisional tandem mass spectrometry. The amino acid sequence of lycocitin 1 was determined as GKLQAFLAKMKEIAAQTL-NH(2). Lycocitin 2 differs from lycocitin 1 by a replacement of a lysine residue for an arginine residue at the second position. Lycocitin 3 differs from the known lycotoxin II consisting of 27 amino acid residues by a deletion of Gly-26. Both lycocitin 1 and 2 inhibit growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Pseudomonas aeruginosa) at micromolar concentrations.     

Structural characterization of novel chemotactic and mastoparan peptides from the venom of the social wasp Agelaiapallipes pallipes by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) techniques were applied for the detection, purification, monitoring, and sequencing of two novel and biologically active peptides occurring at very low levels in the venom of the wasp Agelaia pallipes pallipes. These peptides were sequenced under LC/ESI-MS/MS conditions and designated as Agelaia-CP (I/L-L-G-T-I-L-G-L-L-K-G-I/L-NH2, MW 1207.8 Da) and Agelaia-MP (I/L-N-W-L-K-L-G-K-A-I-I-D-A-I/L-NH2, MW 1565.0 Da). The peptide Agelaia-CP showed no hemolytic activity, but it behaved as a mast cell degranulator and induced a potent chemotaxis in polymorphonucleated leukocyte (PMNL) cells, typical of a wasp chemotactic peptide. The peptide Agelaia-MP showed both powerful mast cell degranulation and hemolysis of washed rat red blood cells, and is thus assigned as a new member of the mastoparan family of peptides. Both peptides seem to be directly involved in the strong inflammatory reactions associated with wasp stings.     

Structural analogues of bradykinin and other vasoactive peptides in the venom of the wasp Polistes gallicus

Six vasoactive peptides have been isolated in the homogeneous state from the venom of the waspPolistes gallicus. Their full amino acid sequences have been determined. It has been shown that four of them are structural analogues of bradykinin with additional N- and C-terminal sequences and intramolecular substitutions of amino acids. Some structure-function relationships in bradykinin analogues are discussed.A. S. Sadykov Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 389–393, May–June, 1990.     

Electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric analyses to solve micro-heterogeneity in post-translationally modified peptides from Phoneutria nigriventer (Aranea, Ctenidae) venom

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.     

Spider venoms: a rich source of acylpolyamines and peptides as new leads for CNS drugs

Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous products from spider venoms, previously explored due to technical limitations. The chemical composition of spider venoms is diverse, ranging from low molecular weight organic compounds such as acylpolyamines to complex peptides. First, acylpolyamines (< 1000 Da) have an aromatic moiety linked to a hydrophilic lateral chain. They were characterized for the first time in spider venoms and are ligand-gated ion channel antagonists, which block mainly postsynaptic glutamate receptors in invertebrate and vertebrate nervous systems. Acylpolyamines represent the vast majority of organic components from the spider venom. Acylpolyamine analogues have proven to suppress hippocampal epileptic discharges. Moreover, acylpolyamines could suppress excitatory postsynaptic currents inducing Ca+ accumulation in neurons leading to protection against a brain ischemic insult. Second, short spider peptides (< 6000 Da) modulate ionic currents in Ca2+, Na+, or K+ voltage-gated ion channels. Such peptides may contain from three to four disulfide bridges. Some spider peptides act specifically to discriminate among Ca2+, Na+, or K+ ion channel subtypes. Their selective affinities for ion channel subfamilies are functional for mapping excitable cells. Furthermore, several of these peptides have proven to hyperpolarize peripheral neurons, which are associated with supplying sensation to the skin and skeletal muscles. Some spider N-type calcium ion channel blockers may be important for the treatment of chronic pain. A special group of spider peptides are the amphipathic and positively charged peptides. Their secondary structure is alpha-helical and they insert into the lipid cell membrane of eukaryotic or prokaryotic cells leading to the formation of pores and subsequently depolarizing the cell membrane. Acylpolyamines and peptides from spider venoms represent an interesting source of molecules for the design of novel pharmaceutical drugs.     

Fast analysis of low molecular mass compounds present in snake venom: identification of ten new pyroglutamate-containing peptides

Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.     

New pharmacological insights of Galactia glaucescens

Galactia glaucescens leaves are popularly used against snakebites in Brazil. The hydroethanolic extract from aerial parts of G. glaucescens (HEGg) was assayed against the neurotoxicity and myotoxicity induced by Bothrops jararacussu venom. A traditional myographic technique was applied for neurotoxicity and the resulting muscles were treated routinely by light microscopy analysis for myotoxicity. Additionally, the antimicrobial potential of HEGg was evaluated against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains, as well as Rutin was isolated for the first time in this specie using chromatographic and spectroscopic methods and its antiophidian property was assessed. HEGg totally prevents the neurotoxicity and myotoxicity effects caused by B. jararacussu, but did not show any antimicrobial effect. Concluding, HEGg and Rutin were able to counteract the toxic effects of the venom and confirmed the antiophidian potential, but not antimicrobial, of G. glaucescens as an alternative for neutralization of B. jararacussu venom.     

Evaluation of biological activities of some Schiff bases and metal complexes

Several Schiff bases were synthesised from sulphonamide and resacetophenone. The characterisation was done by CHN analysis, IR and NMR spectral data. These Schiff bases were evaluated for their antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. The antibacterial activity was studied against B. megaterium, E. coli, B. subtilis, P. fluorescens and antifungal activity against A. awamori. In addition, copper, nickel, cobalt, and iron complexes of two Schiff bases were also synthesised. Their structural characterisation was performed using CHN analysis and IR spectral data and their antibacterial and antifungal activities were also evaluated. The comparison of antimicrobial activities of the ligands and complexes shows that the presence of metal causes more inhibition i.e., more activity. Out of the four metals studied, cobalt and iron were found to have more antimicrobial activity.     

Peptide fingerprinting of snake venoms by direct infusion nano-electrospray ionization mass spectrometry: potential use in venom identification and taxonomy

Fingerprinting by mass spectrometry has been increasingly used to study venom variations and for taxonomic analyses based on venom components. Most of these studies have concentrated on components heavier than 3 kDa, but Bothrops snake venoms contain many biologically active peptides, principally C-type natriuretic peptides and bradykinin-potentiating peptides (BPPs). In this work, we have examined the peptide profile of Bothrops venoms (B. alternatus, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B. leucurus and B. moojeni) using direct infusion nano-electrospray ionization mass spectrometry (nano-ESI-MS) subjecting the data further to principal components analysis (PCA) to assess whether the peptide distributions are reliable in distinguishing the venoms. ESI-MS of a low molar mass fraction obtained by ultrafiltration of each venom (5 kDa nominal cutoff filters) revealed that the venoms have a variety of peptides in common but that each venom also contains taxonomic marker peptides not shared with other venoms. One BPP peptide, QGGWPRPGPEIPP, was found to be common to the seven Bothrops species examined. This peptide may represent a specific marker for this genus since it was not found in the venom of the South American rattlesnake, Crotalus durissus terrificus. PCA on the ESI-MS data reveals a close relationship between B. jararaca, B. jararacussu and B. moojeni venoms, with B. leucurus and B. erythromelas being more distant from these three; B. alternatus and B. insularis were also located distant from these five species, as was C. d. terrificus. These results agree partially with established phylogenetic relationships among these species and suggest that ESI-MS peptide fingerprinting of snake venoms coupled with PCA is a useful tool for identifying venoms and for taxonomic analyses.     

A fast,reproducible and low‐cost method for sequence deconvolution of ‘on‐bead’ peptides via ‘on‐target’ maldi‐TOF/TOF mass spectrometry

A novel approach to high‐throughput sequence deconvolution of on‐bead small peptides (MW < 2000 Da) using on‐target MALDI‐TOF/TOF instrumentation is presented. Short peptides of pentamer and octamer length, covalently attached to TentaGel polystyrene beads through a photolabile linker, were placed onto the MALDI target, apportioned with suitable matrix (2,5‐dihydroxybenzoic acid) and then hit with the instrument laser (Nd : YAG, 355 nm). This induced easy and highly reproducible photochemical cleavage, desorption (MS mode) and fragmentation (MS/MS mode). Peptide fragments were identified with a mass accuracy of 0.1 Da of the expected values. This technique significantly accelerates the sequence determination of positive peptide hits obtained from random combinatorial libraries when screening against biological targets, paving the way for a rapid and efficient method to identify molecular imaging ligands specific to pathological targets in cancer and other diseases. Copyright © 2009 John Wiley & Sons, Ltd.     

Host defense peptides and lipopeptides: modes of action and potential candidates for the treatment of bacterial and fungal infections

Endogenous peptide antibiotics (termed also host-defense or antimicrobial peptides) are known as evolutionarily old components of innate immunity. They were found initially in invertebrates, but later on also in vertebrates, including humans. This secondary, chemical immune system provides organisms with a repertoire of small peptides that act against invasion (for both offensive and defensive purposes) by occasional and obligate pathogens. Each antimicrobial peptide has a broad but not identical spectrum of antimicrobial activity, predominantly against bacteria, providing the host maximum coverage against a rather broad spectrum of microbial organisms. Many of these peptides interact with the target cell membranes and increase their permeability, which results in cell lysis. A second important family includes lipopeptides. They are produced in bacteria and fungi during cultivation on various carbon sources, and possess a strong antifungal activity. Unfortunately, native lipopeptides are non-cell selective and therefore extremely toxic to mammalian cells. Whereas extensive studies have emerged on the requirements for a peptide to be antibacterial, very little is known concerning the parameters that contribute to antifungal activity. This review summarizes recent studies aimed to understand how antimicrobial peptides and lipopeptides select their target cell. This includes a new group of lipopeptides highly potent against pathogenic fungi and yeast. They are composed of inert cationic peptides conjugated to aliphatic acids with different lengths. Deep understanding of the molecular mechanisms underlying the differential cells specificity of these families of host defense molecule is required to meet the challenges imposed by the life-threatening infections.     

Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group

Enterotoxins produced by different species of the Bacillus cereus group, such as cytotoxin K1 (CytK1) and non-haemolytic enterotoxin (NHE), have been associated with diarrhoeal food poisoning incidents. Detection of CytK1 is not possible with commercial assays while NHE is recognised by an immunological kit (TECRA) that does not specifically target this protein because it is based on polyclonal antibodies. It is evident that the lack of suitable tools for the study of enterotoxins hampers the possibilities for accurate hazard identification and characterisation in microbial food safety risk assessment. We applied matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for the detection of CytK1 and NHE produced by pathogenic strains of the B. cereus group using protein digests from 1D gel electrophoresis. Secretion of CytK1 and two of the three components of NHE was confirmed in supernatants of different B. cereus cultures. For each protein, we introduce biomarkers that could be used for the screening of food poisoning or food/environmental isolates that can secrete enterotoxins. For example, tryptic peptides of 2,310.2 and 1,192.5?Da (calculated mass) can be indicators for CytK1 and NheA, respectively, although a simultaneous detection of other enterotoxin-specific peptides is recommended to assure the presence of a toxin in an unknown sample. Comparison of MALDI-TOF/MS with the TECRA kit showed that our methodological strategy performed well and it had the competitive advantage of specifically detecting NheA. Therefore, MALDI-TOF/MS can be successfully incorporated into risk assessment procedures in order to determine the involvement of strains of the B. cereus group in foodborne outbreaks, including the recently described cytK1 producing species, Bacillus cytotoxicus.     

Peptide and glycopeptide dendrimer apple trees as enzyme models and for biomedical applications

Solid phase peptide synthesis (SPPS) provides peptides with a dendritic topology when diamino acids are introduced in the sequences. Peptide dendrimers with one to three amino acids between branches can be prepared with up to 38 amino acids (MW ~ 5,000 Da). Larger peptide dendrimers (MW ~ 30,000) were obtained by a multivalent chloroacetyl cysteine (ClAc) ligation. Structural studies of peptide dendrimers by CD, FT-IR, NMR and molecular dynamics reveal molten globule states containing up to 50% of α-helix. Esterase and aldolase peptide dendrimers displaying dendritic effects and enzyme kinetics (k(cat)/k(uncat) ~ 10(5)) were designed or discovered by screening large combinatorial libraries. Strong ligands for Pseudomonas aeruginosa lectins LecA and LecB able to inhibit biofilm formation were obtained with glycopeptide dendrimers. Efficient ligands for cobalamin, cytotoxic colchicine conjugates and antimicrobial peptide dendrimers were also developed showing the versatility of dendritic peptides. Complementing the multivalency, the amino acid composition of the dendrimers strongly influenced the catalytic or biological activity obtained demonstrating the importance of the "apple tree" configuration for protein-like function in peptide dendrimers.     

Coupled Chromatography for Assay of the Venom Proteome of the Snake Agkistrodon acutus: An Effective Strategy for Discovery of Active Components

Two-dimensional liquid chromatography (2D-LC) is an attractive option for proteome profiling because of its advantages in separation and analysis of proteins and peptides of extreme pH and molecular weight. Proteomics, regarded as a promising and high-throughput method for discovery of new active natural products, calls for technical progress in comprehensive separation, especially for peptides, glycoproteins, and hydrophobic proteins, etc. Here, an optimized off-line IEX-RP LC system has been used for separation of all the components of the venom of the five-pace snake (Agkistrodon acutus). Seventy-nine different natural venom components were obtained by use of this system, more than were obtained by two dimensional electrophoresis of the venom. An automated on-line IEX-RP LC system was also developed for heart-cut separation of components of interest. As a result, we discovered a new tripeptide (PGlu-Asn-Trp, MW 429.1) in the venom. This emphasizes the role of 2D-LC in drug discovery.     

Syntheses of antibacterial peptides,gramicidin S analogs and designed amphiphilic oligopeptides

In order to clarify the relationship between antimicrobial activity and peptide-structure, gramicidin S analogs and cationic α-helical model peptides were designed and synthesized. Introduction of cationic side chains in hydrophilic side of gramicidin S increased antimicrobial activity against Gram-negative bacteria. Amphiphilic structures of the α-helical peptides were found to be effective to show antimicrobial activities against Gram positive bacteria. Increase in number of cationic amino acid residues in the α-helical peptides caused appreciable antimicrobial activities against Gram-negative bacteria, however, induced lower activities against Gram-positive ones.