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See detailMethodology to fish peptide ligands of nAChRs from Cone snail venoms by MALDI-TOF mass spectrometry
Echterbille, Julien ULg; 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 ULg; 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 ULg; Haler, Jean ULg; 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 ULg; Degueldre, Michel ULg; Boulanger, Madeleine ULg 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 ULg; Delvaux, Cédric ULg; Far, Johann ULg 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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See detailExpression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme
Boldrini-Franca, Johara; Santos Rodrigues, Renata; Santos-Silva, Ludier Kesser et al

in Applied Microbiology and Biotechnology (2015), 99

Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous ... [more ▼]

Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar kcat/Km values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications. [less ▲]

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See detailMass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors
Cologna Takeno, Camila; Gilles, Nicolas; Echterbille, Julien ULg et al

in Analytical and Bioanalytical Chemistry (2015), 407

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited ... [more ▼]

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF–TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs [less ▲]

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See detailCombined use of Ion Mobility and Collision-Induced Dissociation to investigate the opening of disulfide bridges by Electron-Transfer Dissociation in peptides bearing two disulfide bonds
Massonnet, Philippe ULg; Upert, Gregory; Smargiasso, Nicolas ULg et al

in Analytical Chemistry (2015)

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability towards enzymatic degradations, and provide the structure and (consequently ... [more ▼]

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability towards enzymatic degradations, and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron Transfer Dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides , resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion-mobility and, (iii) to characterize the species using CID. Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH• radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process. [less ▲]

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See detailAdvances in proteomics for the FP7 Venomics project
Degueldre, Michel ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg

Conference (2014, December 03)

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See detailDevelopment of a high throughput de novo sequencing platform for peptidic toxins combining proteomics and transcriptomics
Degueldre, Michel ULg; Verdenaud, Marion; Zuniga, Sheila et al

Poster (2014, November 07)

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See detailIon mobility-mass spectrometry to perform structural classifications of disulfide-bridged-peptides
Massonnet, Philippe ULg; Upert, Gregory; Morsa, Denis ULg et al

Poster (2014, November)

Detailed reference viewed: 62 (28 ULg)