Abstract :
[en] More than fifty thousands of venomous species, such as snakes, spiders, bees or cone snails, 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 G-protein coupled receptors or ion channels. Mass spectrometry is one the main tools used for toxin characterization. After having introduced the topic and in function of the time I will have, the lecture will be divided into three parts:
(i) Capillary electrophoresis (CE) and ion-mobility (IM) coupled to mass spectrometry as emerging tools for studying the conformation and the fold of small toxins.
Disulfide bonds between cysteine residues are post translational modifications playing essential roles in the biological activity and the stability of numerous peptides or proteins. Intra-molecular disulfide bonds are found in various natural-occurring peptides, and especially in animal toxins. In such peptides, the appropriate cysteine connectivity is mandatory before their chemical synthesis to ensure the bioactivity of the homologue, by providing the perfect peptide conformation. Disulfide bonds connectivity characterization is still a challenging key issue in the analysis of peptides/proteins targeting pharmaceutical or pharmacological utilizations. Thus, the use of sensitive, robust and efficient characterization techniques to access the cysteine pairing is crucial. This study describes the development of new and fast in-solution (CE) and gas-phase (IMMS) methods coupled to mass spectrometry to characterize disulfide bond connectivities of small toxins bearing two disulfide bonds.
(ii) Discovery and mass-spectrometry characterization of Mambaquaretin-1, a peptide from Dendroaspis angusticeps venom that targets type-2 vasopressin receptor against polycystic kidney disease.
Venoms of mambas are mainly composed of large peptide toxins (6-9kDa) targeting a huge diversity of receptors. Even if this property makes them top candidates for the development of innovative molecular tools, their sequencing is far from being trivial. With the decline of Edman sequencers and the continuous increase of mass spectrometry efficacy, it appears interesting to develop a new way to sequence high mass toxins, and particularly those present in tiny quantities in the venom. This work described a based on MALDI-“In-Source Decay”-TOF mass spectrometry (MALDI-ISD-TOF MS) and “bottom-up”. The usefulness of the strategy is demonstrated with the sequencing of Mambaquaretin-1, a Kunitz-fold peptide of 57 aminoacids, representing a novel promising therapeutic agent against polycystic kidney diseases (PKDs). PKDs are a group of genetic disorders causing renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. Mambaquaretin-1 potently and selectively antagonizes three majors V2R activation pathways is described.
(iii) Discovery and characterization of EIIb, a new -conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry.
nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was applied to the screening of Conus ermineus crude venom, aiming at discovering new nAChRs ligands from this venom. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EIIB. The Ki value of this peptide for Torpedo nicotinic receptors was measured at 2.2 +/- 0.7 nM.
