References of "Gilles, Nicolas"
     in
Bookmark and Share    
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 43 (8 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 21 (3 ULg)
Full Text
Peer Reviewed
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)

Detailed reference viewed: 44 (24 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 33 (9 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 72 (13 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 80 (38 ULg)
Full Text
Peer Reviewed
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)

Detailed reference viewed: 35 (3 ULg)
Full Text
Peer Reviewed
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: 32 (10 ULg)
Full Text
Peer Reviewed
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: 67 (32 ULg)
Full Text
Peer Reviewed
See detailNew mass spectrometry based methodology to sequence a whole snake venom
Echterbille, Julien ULg; Boulanger, Madeleine; Degueldre, Michel ULg et al

Poster (2014, February 10)

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 sequencing, and especially when using 1,5-DAN as reducing matrix. 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 ▲]

Detailed reference viewed: 117 (24 ULg)
See detailAdvances in proteomics for the FP7 Venomics project - Disulfide bridge assignement task
Massonnet, Philippe ULg; Upert, Gregory; Pastor, Alexandra et al

Scientific conference (2013, December 18)

Detailed reference viewed: 38 (12 ULg)
Full Text
Peer Reviewed
See detailAttribution of Cysteine Connectivities in small toxins - New Prospects Based on Partial Oxidation/Reduction Experiments and Ion-Mobility Mass Spectrometry
Quinton, Loïc ULg; Massonnet, Philippe ULg; Echterbille, Julien ULg et al

Conference (2013, December)

Disulfide bonds are post-translational modifications often found in biological compounds and especially in animal toxins. Disulfide bonds participate in the formation of specific folding of peptides and ... [more ▼]

Disulfide bonds are post-translational modifications often found in biological compounds and especially in animal toxins. Disulfide bonds participate in the formation of specific folding of peptides and proteins, directly related to their biological activity. Cystein pairing determinations are primordial for the synthesis of chemical homologous displaying the same bioactivity than the natural compound. This task appears already difficult when the cysteine pairings have to be determined from large proteins. The combination of physical and chemical techniques such as NMR, enzymatic proteolysis, liquid chromatography and mass spectrometry, is needed to circumvent this difficulty. However, when the work concerns small compounds such as conotoxins, the problem is much more complex due to the low amount of available compound and to the lack of enzymatic cleavage sites between cysteines. In this study, we investigate the case of small peptides that contain two disulphide bonds. The idea is to determine the cystein pairings in such compounds by a chemical partial reduction (or oxidation) of the peptides, followed by the separation of the generated species by ion-mobility mass spectrometry, and their characterisation by tandem mass spectrometry. Up to now, we have investigated the partial reduction not only in solution (with DTT and TCEP) but also in the gas-phase (Electron transfer dissociation), and partial oxidation in solution (with 3-CPBA). The results demonstrate an unexpected complexity of the data, including low fragmentation ratios of peptides and disulfide scramblings. [less ▲]

Detailed reference viewed: 58 (15 ULg)