|Reference : New Methodology to detect toxin-GPCR binding by MALDI-TOF Mass Spectrometry|
|Scientific congresses and symposiums : Poster|
|Physical, chemical, mathematical & earth Sciences : Chemistry|
|New Methodology to detect toxin-GPCR binding by MALDI-TOF Mass Spectrometry|
|[en] Nouvelle méthodologie pour détecter la liaison toxine-RCPG par spectrométrie de masse MALDI-TOF|
|Echterbille, Julien [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|De Pauw, Edwin [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|Gilles, Nicolas [> >]|
|Quinton, Loïc [Université de Liège - ULg > Département de chimie (sciences) > Chimie biologique >]|
|59th Conference on Mass Spectrometry and Allied Topics|
|du 5 juin 2011 au 9 juin 2011|
|American Society for Mass Spectrometry|
|[en] GPCR ; MALDI-TOF ; Venomics|
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.
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.
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.
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).
|Laboratoire de Spectrométrie de Masse|
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