References of "Quinton, Loïc"
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See detailSeparation, identification and quantification of peptidoglycan fragments by zwitterionic hydrophilic interaction chromatography and capillary electrophoresis coupled to mass spectrometry
Boulanger, Madeleine ULiege; Delvaux, Cédric ULiege; Raymackers, Alice ULiege et al

Poster (2017, June 05)

Bacterial peptidoglycan-derived muropeptides and peptides are soluble fragments acting as messengers in diverse cell-signalling events. As the peptidoglycan wall is a key target of antibiotics, bacteria ... [more ▼]

Bacterial peptidoglycan-derived muropeptides and peptides are soluble fragments acting as messengers in diverse cell-signalling events. As the peptidoglycan wall is a key target of antibiotics, bacteria have developed specific resistance mechanisms based on the detection of these fragments inside their cytoplasm. In our model strain, Bacillus licheniformis, the peptidoglycan dipeptide m-A2pm-D-Glu triggers a beta-lactamase induction. However, the nature and the concentration of cytoplasmic peptidoglycan fragments leading to the dipeptide formation are unknown. Additionally, the muropeptides sensing is involved in the innate immune response toward bacterial invasion and is therefore of considerable importance in eukaryotes self-defence functions. In this context, the development of reliable analytical methods aiming to identify and quantify those fragments in complex samples are of major interest. [less ▲]

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See detailGreen mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease
Ciolek, Justyna; Reinfrank, Helen; Quinton, Loïc ULiege et al

in Proceedings of the National Academy of Sciences of the United States of America (2017)

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin ... [more ▼]

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein–coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 μ𝝁g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs. [less ▲]

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See detailDiscovery and characterization of EIIB, a new α-conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry
Echterbille, Julien; Gilles, Nicolas; Araoz, Romulo et al

in Toxicon (2017), 130

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α ... [more ▼]

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EIIB. The Ki value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM. [less ▲]

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See detailDiversity in sequences, post-translational modifications and expected pharmacological activities of toxins from four Conus species revealed by the combination of cutting-edge proteomics, transcriptomics and bioinformatics
Degueldre, Michel; Verdenaud, Marion; Garikoitz, Legarda et al

in Toxicon (2017), 130

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top ... [more ▼]

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top candidates for the development of innovative drugs. Instead of relying on the classical, low-throughput bioassay-guided approach to identify innovative bioactive peptides, this work exploits a recent paradigm to access to venom diversity. This strategy bypasses the classical approach by combining high-throughput transcriptomics, proteomics and bioinformatics cutting-edge technologies to generate reliable peptide sequences. The strategy employed to generate hundreds of reliable sequences from Conus venoms is deeply described. The study led to the discovery of (i) conotoxins that belong to known pharmacological families targeting various GPCRs or ion-gated channels, and (ii) new families of conotoxins, never described to date. It also focusses on the diversity of genes, sequences, folds, and PTM's provided by such species. [less ▲]

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See detailPeptidoglycan fragments separation and identification by zwitterionic hydrophilic interaction chromatography and capillary electrophoresis coupled to mass spectrometry
Boulanger, Madeleine ULiege; Raymackers, Alice ULiege; Delvaux, Cédric ULiege et al

Poster (2017, February 08)

Bacterial peptidoglycan-derived peptides and muropeptides are soluble unique fragments acting as messengers in diverse cell-signalling events. As the bacterial peptidoglycan wall is a major target of ... [more ▼]

Bacterial peptidoglycan-derived peptides and muropeptides are soluble unique fragments acting as messengers in diverse cell-signalling events. As the bacterial peptidoglycan wall is a major target of antibiotics, bacteria have developed specific resistance mechanisms based on the detection of such fragments. In addition, the muropeptides sensing is involved in the innate immune response toward bacterial invasion and is therefore of major importance in the eukaryotes self-defence functions. In Bacillus licheniformis 749/I, the peptidoglycan dipeptide m-A2pm-D-Glu triggers beta-lactam resistance via the induction of a beta-lactamase, BlaP. This induction process relies on a complex regulation system for which the nature and the concentration of peptidoglycan fragments leading to the formation of dipeptide moiety inside the cytoplasm are unknown. In this context, the development and the validation of a reliable method to identify and quantify those cytoplasmic fragments is of major interest. Conventionally, the peptidoglycan is first digested by mutanolysin in order to generate muropeptides which are subsequently analyzed by reversed-phase liquid chromatography (RP-LC, C18). However, this technique is not effective enough to separate the peptides that, as a result, are eluted in the flow through . In this work, we developed two novel analytical separation methods, namely capillary electrophoresis (CE) and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) both coupled to mass spectrometry (MS), aiming at overcoming the drawbacks encountered in traditional separation techniques. Both methods show great results in the identification of peptidoglycan fragments in complex samples. CE analysis lead to muropeptides and peptides separation whereas ZIC-HILIC only retains peptides. Nevertheless, the latter has been optimized and validated for the cytoplasmic peptidoglycan peptides identification and quantification. Althogether, ZIC-HILIC-MS and CE-MS have proved to be powerful analytical tools for the identification and quantification of peptidoglycan fragments in complex matrix samples. Further optimizations are still ongoing for the analysis of muropeptides, which hopefully will lead to the identification and quantification of cytoplasmic peptidoglycan fragments composition during the Bacillus licheniformis 749/I BlaP beta-lactamase induction process. [less ▲]

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See detailHigh-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery
Turchetto; Sequeira, Ana Filipa; Ramond, Laurie et al

in Microbial Cell Factories (2017), 16(6), 1-15

Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane ... [more ▼]

Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. RESULTS: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. CONCLUSIONS: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs. [less ▲]

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See detailContribution of Cross-linking and Ion-mobility for the study of protein and complex structures
Baumans, France ULiege; Grifnée, Elodie ULiege; Hage, Christoph et al

Poster (2016, September 05)

The tridimensional structures of proteins and the mapping of protein-protein interactions are precious sources of information for the understanding of their function. Different techniques such as X-ray ... [more ▼]

The tridimensional structures of proteins and the mapping of protein-protein interactions are precious sources of information for the understanding of their function. Different techniques such as X-ray cristallography or nuclear magnetic resonance are usually used to achieve this goal. In the field of mass spectrometry, several tools were also developped. The one presented here is the chemical cross-linking in which two reactive residue side chains, spatially close, are linked thanks to a bifunctional chemical, called crosslinker. Ion-mobility coupled to mass spectrometry has also been investigated for the study of cross-linked products. The first results tend to show that cross-linkers allow to fix the shape of the protein in solution, leaving it intact when analysed in the gas phase. [less ▲]

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See detailFirst Results Using TIMS on Systems Requiring High IMS Resolution
Haler, Jean ULiege; Massonnet, Philippe ULiege; Morsa, Denis ULiege et al

Conference (2016, June 05)

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See detailMethodology to fish peptide ligands of nAChRs from Cone snail venoms by MALDI-TOF mass spectrometry
Echterbille, Julien ULiege; Gilles, Nicolas; Araoz, Romulo et al

Poster (2016, June)

More than 50,000 of venomous species are currently indexed in the world. Each of their venom is composed of hundreds of toxins which potentially exhibit a high selectivity for membrane receptors such as ... [more ▼]

More than 50,000 of venomous species are currently indexed in the world. Each of their venom is composed of hundreds of toxins which potentially exhibit a high selectivity for membrane receptors such as GPCRs or ion channels. Among them, nAChRs are a target for drug discovery, primarily for treating central nervous system troubles. Therefore, the discovery of pharmacological tools and innovative drugs targeting nAChRs from animal venoms appears as an evidence. This study proposes the use a mass-spectrometry based methodology1 to discover new nAChRs ligands from cone snails venoms, and particularly -conotoxins (a-CTXs), known as potential antagonists of nAChRs2. in few words, Torpedo membranes, containing a high concentration of nAChRs, are incubated with BSA tryptic digests (>100 peptides) doped by small amounts of known a-CTXs. After two hours incubation, free (i.e. containing molecules remaining in solution) and bound (i.e. peptides bound to the membranes) fractions were analyzed with a MALDI-TOF/TOF mass spectrometer. The POC (positive and negative controls) as well as a real screening of Conus ermineus venom are presented. [less ▲]

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See detailNew methodology to detect toxin-nAChRs binding by MALDI-TOF mass spectrometry
Echterbille, Julien ULiege; Gilles, Nicolas; Araoz, Romulo et al

Conference (2016, April 18)

More than 50 thousands of venomous species are currently indexed in the world. Each of their venoms is composed of 200 to 1000 different toxins which potentially exhibit a high selectivity for membrane ... [more ▼]

More than 50 thousands of venomous species are currently indexed in the world. Each of their venoms is composed of 200 to 1000 different toxins which potentially exhibit a high selectivity for membrane receptors such as G-protein coupled receptors or ion channels such nicotinic acetylcholine receptors (nAChRs). The latter have been a target for drug discovery efforts, primarily for central nervous system indications. Therefore, it appears of prime interest to design new pharmacological tools and potentially discover future drugs targeting this kind of ion channels. In 2015, our group published a new mass spectrometry-based methodology to screen peptide ligands for G protein coupled receptors1. The proof of concept of this methodology was built by studying the binding of [Arg8]-vasopressin (AVP) on type 2-vasopressin receptor (V2). We extended this methodology to another system ligand-receptor. As all Conus species venoms investigated so far contain at least one toxin antagonizing nAChRs: the alpha-conotoxins. Therefore, the ligand-receptor model couple that has been chosen is nAChRs-alpha-conotoxins. Experimentally, fragments of cellular membranes over-expressing nAChRs were incubated with Bovine Serum Albumine (BSA) tryptic digest (~100 peptide toxins) doped by a small amount of Alpha-conotoxins. After 2 hours incubation, free and bound fractions were purified with a combination of centrifugation and micro column purifications. Samples were finally analyzed with a MALDI-TOF/TOF mass spectrometer. By comparison of the intensity of Alpha-conotoxins in the free and in the bound fractions, we clearly detect an enrichment of nAChRs ligand in the latter. In order to transpose the methodology to natural mixture, we applied the workflow to crude conus venoms. We incubated membranes over-expressing nAChRs with Conus textile venom which is known to possess at least 5 different alpha-conotoxins. Thanks to our approach, we were able to detect an enrichment of these known ligands in the bound fraction. In order to validate the potential of our approach, the next step of this work will be the incubation of a Conus venom for which no alpha-conotoxins have been described. [less ▲]

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See detailIon Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s)
Massonnet, Philippe ULiege; Haler, Jean ULiege; Upert, Gregory et al

in Journal of the American Society for Mass Spectrometry (2016)

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See detailIsolation and characterization of Ts19 Fragment II, a new long-chainpotassium channel toxin from Tityus serrulatus venom
Cerni, Felipe Augusto; Pucca, Manuela Berto; Amorim, Fernanda Gobbi et al

in Peptides (2016), 80

Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is aprotein presenting 49 amino acid residues, three disulfide bridges, Mr5534 Da and was ... [more ▼]

Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is aprotein presenting 49 amino acid residues, three disulfide bridges, Mr5534 Da and was classified as a newmember of class (subfamily) 2 of the -KTxs, the second one described for Ts scorpion. The -KTx familyis composed by two-domain peptides: N-terminal helical domain (NHD), with cytolytic activity, and aC-terminal CS domain (CCD), with Kv blocking activity. The extensive electrophysiological screening(16 Kv channels and 5 Nav channels) showed that Ts19 Frag-II presents a specific and significant blockingeffect on Kv1.2 (IC50value of 544 ± 32 nM). However, no cytolytic activity was observed with this toxin.We conclude that the absence of 9 amino acid residues from the N-terminal sequence (compared to Ts19Frag-I) is responsible for the absence of cytolytic activity. In order to prove this hypothesis, we synthesizedthe peptide with these 9 amino acid residues, called Ts19 Frag-III. As expected, Ts19 Frag-III showed tobe cytolytic and did not block the Kv1.2 channel. The post-translational modifications of Ts19 and itsfragments (I–III) are also discussed here. A mechanism of post-translational processing (post-splitting) issuggested to explain Ts19 fragments production. In addition to the discovery of this new toxin, this reportprovides further evidence for the existence of several compounds in the scorpion venom contributing tothe diversity of the venom arsenal. [less ▲]

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See detailProteomic landscapes of Pachycondila villosa ant venom by nano-scale chromatography and high resolution mass spectrometry
Cologna Takeno, Camila; Degueldre, Michel; Shibao, Priscilla et al

Poster (2016)

Introduction: It is estimated that the total number of molecules present in animal venoms is a collection of 40 million different compounds and, despite the efforts made, less than 0,01% of those ... [more ▼]

Introduction: It is estimated that the total number of molecules present in animal venoms is a collection of 40 million different compounds and, despite the efforts made, less than 0,01% of those compounds was identified and characterized to date. However, recent progresses in proteomic, in parallel with the advances of mass spectrometry have contributed to the study of those bio-libraries. The sensitivity improvement of these instruments allows the study of minimal amounts of sample still yielding a wealth of information. The present work aimed to perform a deep proteomic analysis of the venom from the ant Pachycondilyla villosa focusing on the de novo sequencing and the characterization of post translational modifications using high resolution mass spectrometers. Methods: The crude venom (0,5 ug) of P. villosa ants collected on Panga Natural Reserve (Uberlandia-Minas Gerais- Brazil) was diluted in 0,2% of formic acid and injected into a nanoACQUITY ULPC equipped with a monolithic PepSwift Capillary column 100µm x 25, hyphened to a Q Exactive Orbitrap mass spectrometer. The elution of the compounds was performed with a gradient of 3 to 50% of solution B in 80 minutes (A: H2O/FA 0.1%; B: ACN) at flow rate of 1 µL/min. All mass spectrometry analyses were performed in data dependent analysis (DDA) mode that automatically triggers the MS/MS experiments. The top 10 most intense peaks of each MS scan was fragmented by high-energy dissociation (HCD) and their corresponding MS/MS spectra were acquired. Preliminary data: Animal venoms are considered a rich source of biologically active compounds, which has been constantly selected and refined by the processes of natural evolution, in which each molecule is endowed with pharmacological properties highly valuable for scientific purposes. Despite the commitment, the exploration of these bio-libraries remains limited which might be related to the technological limitations that prevent full-scale investigation of these venoms. In addition, the conventional methods used to explore animal venoms are still time-consuming and require large amounts of samples, which restrict the studies for a few species. Unquestionably, the advances of proteomics and mass spectrometry instrumentations benefited a great deal the research on hymenoptera venom. Mostly due to their small size and therefore scarcely collected venom, this order has always been neglected and considered unfeasible to be studied through the known strategies. The present work represents the first report concerning the venom composition of P. villosa ant. The preliminary results already highlight the complexity of this venom, which showed to be composed by over 5000 different molecules. Most of those components fall into the 800- 4000 Da range, which is in agreement with other studies regarding ant´s venom composition. Most of the proteomics studies concerning ant venoms already revealed the presence of linear peptides below 5000 Da as major components. Those small peptides usually display antimicrobial activity and some of them hold additional insecticidal activity. Novel aspect: The results obtained already point out the biotechnological potential of P. villosa venom and highlight’s its complexity [less ▲]

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See detailHigh-throughput sequencing of toxins with pharmacological interest: proof of concept and first applications
Echterbille, Julien ULiege; Degueldre, Michel ULiege; Boulanger, Madeleine ULiege et al

Conference (2015, September 28)

Animal venoms are complex chemical cocktails, comprising wide ranges of biologically active reticulated peptides that target with high selectivity and efficacy varieties of membrane receptors. Assuming ... [more ▼]

Animal venoms are complex chemical cocktails, comprising wide ranges of biologically active reticulated peptides that target with high selectivity and efficacy varieties of membrane receptors. Assuming the fact that each of the 170,000 venomous species reported can produce more than 250 bioactive toxins, at least 40,000,000 bioactive peptides and proteins may be discovered. Among the four described species of mambas, Eastern Jameson’s mamba (Dendroaspis jamesonii kaimosae) venom is the less characterized since only 9 peptides are referenced in database. This work aims at developing a new strategy devoted to the deep analysis of animal venoms. Our approach consists in a first separation of the venom using cation exchange chromatography. Each primary fraction is then purified a second time by classical RP-HPLC. A total of 328 fractions, containing amongst 1 and 4 toxins, are finally collected. MALDI-MS analysis of each fraction is done in order (1) to obtain information about masses and (2) to obtain sequences of toxins thanks to MALDI-In Source Decay (ISD) dissociation coupled with on MALDI target plate reduction of the peptides. ISD has already been demonstrated efficient for toxin sequencing1, and especially when using 1,5-DAN as reducing matrix2. ISD yields to sequences that cover more than 50% of peptide sequences by series of singly charged c-type ions. Thanks to this methodology, we were able to obtain 85% of satisfactory results i.e. spectra giving quite long tags of amino acids (up to 20 residues). As a way to validate our method, a tag coming from ISD spectrum interpretation has found a match in database for an Eastern Jameson’s mamba toxin. The global sequence has then been obtained by extrapolation on the ISD spectrum. Since ISD spectra are simpler than classical MS/MS spectra, automation of spectra interpretation, difficult with other fragmentation techniques (CID, ETD…), is implementable. In the near future, sequences obtained with this approach will be used to direct tests of biological activity through sequence homologies with already known ligands for different kinds of membrane receptors. [less ▲]

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See detailCharacterization of Venom Peptides using Microfluidic Separation Techniques coupled to Mass Spectrometry (LC-MS and CE-MS)
Degueldre, Michel ULiege; Delvaux, Cédric ULiege; Far, Johann ULiege et al

Poster (2015, March)

More than the half of the principal sub-kingdoms of the animal world contains species that produce venom whose purposes are to immobilize, kill and pre-digest the preys. These venoms represent an ... [more ▼]

More than the half of the principal sub-kingdoms of the animal world contains species that produce venom whose purposes are to immobilize, kill and pre-digest the preys. These venoms represent an exceptionally rich source of various biologically active peptides, both in their structures and their effects, which are more and more useful for human being1. Yet, the total characterization of such complex samples require advanced analytical techniques mainly due to the complexity of the sample (hundreds of compounds), the limited quantities usually available and the presence of numerous PTMs, especially disulfide bridges and specific folding. Here we present a method that combines LC and CE separation techniques coupled to mass spectrometry (MS) to characterize the peptide composition of the snake venom Naja atra. The characterization will not only focus on the toxin sequencing (LC-MS and LC-MS/MS), but will also aim at analyzing the folding of the toxins (CE-MS). To this end, native and reduced/alkylated toxins will be analyzed by both techniques. Final result targets the determination of the global hydrophobic pattern and native tridimensional folding of these strongly reticulated peptides. (1) Richard J. Lewis & Maria L. Garcia, Therapeutic potential of venom peptides. Nature Reviews Drug Discovery 2003, 2, 790-802. [less ▲]

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