|Reference : Disulfide bond scrambling in partially reduced and alkylated peptides revealed by Ion...|
|Scientific congresses and symposiums : Poster|
|Physical, chemical, mathematical & earth Sciences : Chemistry|
|Disulfide bond scrambling in partially reduced and alkylated peptides revealed by Ion Mobility Mass Spectrometry|
|[en] Réarrangement de ponts disulfures dans les peptides partiellement réduit et alkylés révélé par spectrométrie de mobilité ionique et de masse.|
|Echterbille, Julien [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|Quinton, Loïc [Université de Liège - ULg > Département de chimie (sciences) > Chimie biologique >]|
|De Pauw, Edwin [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|NVMS–BSMS International Congress on Mass Spectrometry|
|du 28 mars 2012 au 30 mars 2012|
|[en] disulfide bond ; ion mobility ; mass spectrometry|
|[en] 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.
|Laboratoire de Spectrométrie de Masse|
|Researchers ; Professionals|
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