Les venins d'animaux, nouvelle panacée?

in Athena (2013)

Araignées, serpents, scorpions,… autant d’animaux ayant une place particulière dans l’imaginaire collectif. Fascinants, horripilants voire même terrifiants, les adjectifs ne manquent pas pour qualifier ... [more ▼]

Araignées, serpents, scorpions,… autant d’animaux ayant une place particulière dans l’imaginaire collectif. Fascinants, horripilants voire même terrifiants, les adjectifs ne manquent pas pour qualifier les réactions qu’ils suscitent auprès des populations. Que dire alors de la peur engendrée par leur venin. Une simple piqûre, morsure ou contact peut s’avérer extrêmement dangereux voire létal... [less ▲]

Disulfide bond scrambling in partially reduced and alkylated peptides revealed by Ion Mobility Mass Spectrometry

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 ▲]

Disulfide bond assignement and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry

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 ▲]

Disulfide bonds assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry

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 ▲]

Disulfide bond assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry

Poster (2011)

MALDI-TOF/TOF sequencing of peptide toxins from animal venoms

Poster (2010, April 16)