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See detailIon-Mobility mass spectrometry as a potential tool to assign disulfide bonds arrangements in peptides with multiple disulfide bridges.
Echterbille, Julien ULg; Quinton, Loïc ULg; Gilles, Nicolas et al

in Analytical Chemistry (2013)

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually ... [more ▼]

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using MS/MS spectra of the totally reduced unfolded species but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of non-classical ions. MS/MS alone does not allow the cysteine pairing nor the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining Ion Mobility Spectrometry (IMS)and Collision Induced Dissociation(CID). It is assumed that the opening of one S-S bridges in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence, but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteines connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference. [less ▲]

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See detailSecretion and maturation of conotoxins in the venom ducts of Conus textile
Dobson, Rowan ULg; Collodoro, Mike; Gilles, Nicolas et al

in Toxicon (2012), 60(8), 1370-1379

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

Scientific conference (2012, April)

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See detailDisulfide bond scrambling in partially reduced and alkylated peptides revealed by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg

Poster (2012, March 29)

Animal venoms are mainly composed of peptide toxins, which are highly structured by many disulfide bridges. In these toxins, disulfides play different major roles such as increasing the toxins efficiency ... [more ▼]

Animal venoms are mainly composed of peptide toxins, which are highly structured by many disulfide bridges. In these toxins, disulfides play different major roles such as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. Peptide sequencing followed by determination of the cysteine pairings is still challenging and, therefore, an important step in structural analysis. This work was, in its beginning, focused on the development of ion mobility (IMS) based methodology used to assign disulfides. The strategy relies on the analysis of partially reduced/alkylated disulfide containing peptides. The resulting mixture is analyzed by ion mobility, followed by MS/MS acquisition on each mobility resolved species. Surprisingly, first investigations revealed, after partial reduction, a disulfide rearrangement phenomenon. Indeed, some of the cystein pairings were not those expected to be. These experiments were conducted on ¿-CnI and ¿-GI toxins purified from the venoms of Conus consors and Conus geographus marine snails, respectively. Each toxin contains four cysteines linked together with two disulfide bridges. Peptides were partially reduced by an excess of dithiothreitol and then alkylated by a large excess of iodoacetamide. The resulting mixture was purified on a microcolumn before being analyzed by nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Each toxin partially reduced/alkylated results, theoretically, in a mixture of fully oxidized (two disulfides oxidized), fully reduced (two disulfides reduced) and partially reduced forms (one of the two disulfides reduced). Thanks to the mass shift created by the alkylation, an isolation of the species which m/z ratio corresponds to one disulfide reduced and alkylated has been done in the quadrupole before the mobility separation. The arrival time distribution of triply charged ions reveals the presence of different species (4 in the case of ¿-GI and 2 for ¿-CnI), characterized by different relative cross sections in the gas-phase. As ion mobility resolved species give characteristic fragments upon fragmentation (after IMS), we were able to identify a scrambling of the disulfides (isomerization). In simple words, other disulfide bonds than expected ones were characterized. We suppose that the scrambling phenomenon occurs in solution,during the reduction step, since the alkylation cannot avoid rearrangement. The method is now being applied to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailG protein-coupled receptors, an unexploited animal toxin targets: Exploration of green mamba venom for novel drug candidates active against adrenoceptors
Maïga, Arhamatoulaye; Mourier, Gilles; Quinton, Loïc ULg et al

in Toxicon (2012), 59

At a time when pharmaceutical companies are having trouble finding new low MW drugs and when biologics are becoming more common, animal venoms could constitute an underexploited source of novel drug ... [more ▼]

At a time when pharmaceutical companies are having trouble finding new low MW drugs and when biologics are becoming more common, animal venoms could constitute an underexploited source of novel drug candidates. We looked for identifying novel animal toxins active against G protein-coupled receptors (GPCR), the most frequently exploited class of treatment targets, with the aim to develop novel research tools and drug candidates. Screening of green mamba (Dendroaspis angusticeps) venom against adrenoceptors identified two novel venom peptides. r-Da1a shown an affinity of 0.35 nM for the a1a-AR while r-Da1b displayed affinities between 14 and 73 nM for the three a2-ARs. These two venom peptides have sequences similar to those of muscarinic toxins and belong to the three-finger-fold protein family. a1a-AR is the primary target for the treatment of prostate hypertrophy. In vitro and in vivo tests demonstrated that r-Da1a reduced prostatic muscle tone as efficiently as tamsulosin (an antagonist presently used), but with fewer cardiovascular side effects. a2-ARs are the prototype of GPCRs not currently used as treatment targets due to a lack of specific ligands. Blockage of these receptors increases intestinal motility, which may be compromised by abdominal surgery and reduces orthosteric hypotension. In vitro and in vivo tests demonstrated that r-Da1b antagonizes a2-ARs in smooth muscles and increased heart rate and blood catecholamine concentrations. These results highlight possible exploitation of r-Da1a and r-Da1b in important pathologies. [less ▲]

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See detailThe usefulness of Ion Mobility-Mass Spectrometry for Small Molecules Analysis
Far, Johann ULg; Goscinny, Séverine ULg; Joly, Laure et al

Conference (2012, March)

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See detailMALDI In-Source Decay for High Throughput sequencing of peptide animal toxins
Quinton, Loïc ULg; Degueldre, Michel ULg; Gilles, Nicolas et al

Poster (2012)

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See detailMass spectrometry as a tool to search specific ligands for G protein coupled receptors.
Cologna, Camila Takeno; Echterbille, Julien ULg; De Pauw, Edwin ULg et al

Conference (2012)

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See detailDu venin toxique à la découverte de nouveaux composés thérapeutiques
Quinton, Loïc ULg

in Bulletin de l'association des chimistes de l'ULg (2012), Bulletins 3 et 4

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

Scientific conference (2012)

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See detailAn Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation
Quinton, Loïc ULg; Gilles, Nicolas; Smargiasso, Nicolas ULg et al

in Journal of the American Society for Mass Spectrometry (2011), 22(11), 1891-1897

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several ... [more ▼]

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of 7 to 25 kDa. Secondly, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about one third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence. The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family of peptides. [less ▲]

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See detailDisulfide bond assignement and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; Rosu, Frédéric ULg et al

Conference (2011, October 11)

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their ... [more ▼]

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in structural analysis. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin’s sequence contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1). The resulting mixture was purified before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Partial reduction of α-CnI results in a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by different relative cross sections in the gas-phase. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M+4Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M+2Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailMass spectrometry as an evolutive tool for toxinology: from sequencing to toxin shapes
Quinton, Loïc ULg; Gilles, Nicolas; Echterbille, Julien ULg et al

Conference (2011, September 12)

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See detailDisulfide bonds assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg et al

Conference (2011, April 29)

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their ... [more ▼]

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in structural analysis. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin’s sequence contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1). The resulting mixture was purified before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Partial reduction of α-CnI results in a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by different relative cross sections in the gas-phase. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M+4Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M+2Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailNew Methodology to detect toxin-GPCR binding by MALDI-TOF Mass Spectrometry
Echterbille, Julien ULg; De Pauw, Edwin ULg; Gilles, Nicolas et al

Poster (2011)

Introduction 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 ... [more ▼]

Introduction 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 ionic channels or G-protein coupled receptors (GPCRs). GPCRs constitute the larger family of receptors since around 800 different kinds of them are knows. GPCRs are the target of around 30% of the current pharmacopeia drugs. Notable examples include Novartis’s Zelnorm, Eli Lilly’s Zyprexa and Schering-Plough’s Clarinex used to treat constipation, schizophrenia and allergies, respectively. Finding new GPCRs ligands appears of prime interest to design new pharmacological tools and potentially discover the drugs of our future. Interestingly, several toxins from venoms have already been described to bind to this particular family of receptor, opening the way to the discovery of new peptide drugs from animal venoms1-2. This work presents a pioneering MALDI-TOF/TOF based strategy to fish new GPCRs ligands from complex mixtures such as venom fractions. Methods The proof of concept of this methodology was built by studying the binding of [Arg8]-vasopressin (AVP) on type 2-vasopressin receptor (V2). Experimentally, fragments of cellular membranes over-expressing V2 receptors were incubated with cone snail’s venom fraction (~30 peptide toxins) doped by a small amount of AVP. After 2 hours incubation, free and bound fractions were carefully purified with a combination of centrifugation and micro column purifications. Samples were finally analyzed with a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer and the resulting spectra were interpreted with FlexAnalysis (v3.0), BioTools (v3.2) and SequenceEditor (v3.2) bioinformatics’ softwares from Bruker Daltonics. Preliminary data After the incubation of cellular membranes overexpressing V2 GPCR with a complex mixture of peptides doped by AVP, we clearly detect that the only V2 ligand present in the fraction was the AVP. Our result demonstrates the possibility to identify a ligand of GPCRs from a complex peptide mixture, such as venom fractions. Contrary to radiobinding, this approach allows detecting the direct binding of the toxin and does not imply to know a ligand of the studied GPCR before starting the experiments. This opens the way to the deorphanization of receptors (180 orphans GPCRs over 800). Moreover, since the new ligand is detected by mass spectrometry, it is directly identified from the mixture, without additional purification. Its structural characterization can be directly performed by de novo sequencing experiments. The drawback of our approach is the very long (but crucial!) sample preparation as each sample requires 2 purification steps (for both free and bound fraction). The next step of our work will be the automation of the procedure to allow a high-throughput screening of venom fractions on different GPCRs and the discovery of new ligands. Novel aspect GPCR’s ligands discovery by MALDI-TOF/TOF based techniques: a new pharmacological tool. 1 Quinton, L. et al. Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the a1A-adrenoceptor. British Journal of Pharmacology 159, 316-325 (2010). 2 Rouget, C. et al. Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors. British Journal of Pharmacology 161, 1361-1374, doi:10.1111/j.1476-5381.2010.00966.x (2010). [less ▲]

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See detailDisulfide bond assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; Rosu, Frédéric ULg et al

Poster (2011)

Introduction Animal venoms are mainly composed of peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges are a key feature as (i) they increase the toxins efficiency ... [more ▼]

Introduction Animal venoms are mainly composed of peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges are a key feature as (i) they increase the toxins efficiency by lowering their immunogenicity; (ii) they provide the adequate conformation for high affinity binding to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in their structure analysis and the understanding of their interactions with receptors. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. Methods The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin sequence is GRCCHPACGKYYSC-NH2. It contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1) at 56°C during 30min. The resulting mixture was purified by ZipTip C18 micro columns before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Mobilograms and mass spectra were analyzed using MassLynx (v4.1) and Driftscope (v2.1) from Waters. Preliminary data Partial reduction of a-CnI was performed in order to obtain a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (only one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by a different relative cross sections in the gas-phase. The charge state of the ions influences the ion mobility separation. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M=1541.58 Da). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M=1545.6 Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M=1543.59 Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. Novel aspect Sequencing and disulfide bridges assignment of peptide toxins using ion mobility resolved MS/MS [less ▲]

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See detailWhat can Mass Spectrometry do for Toxinology?
Quinton, Loïc ULg

Conference (2010, December 10)

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See detailMass spectrometric sequencing of peptidic toxins : an overview
Quinton, Loïc ULg; Echterbille, Julien ULg; Pierre, Escoubas et al

in Editions de la SFET – SFET Editions (2010)

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