Julien Echterbille

University of Liège

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Loïc Quinton

University of Liège

Edwin De Pauw

University of Liège

Nicolas Gilles

Commissariat à l'Énergie Atomique et aux Énergies Alternatives

Michel Degueldre

University of Liège

Denis Servent

Technologies pour la Santé

Eliane Candiani Arantes

Universidade de São Paulo

Rómulo Aráoz

Technologies pour la Santé

Fernanda Gobbi Amorim

University of Liège

Nicolas Smargiasso

University of Liège

Steve Peigneur

KU Leuven

Cooperative Institutions

University of Liège

Universidade de São Paulo

Centre National de la Recherche Scientifique

KU Leuven

Université Paris-Saclay

Commissariat à l'Énergie Atomique et aux Énergies Alternatives

Technologies pour la Santé

CEA Paris-Saclay

Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement

Universidade Vila Velha

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Mass spectrometric sequencing of peptidic toxins : an overview

Loïc Quinton Julien Echterbille Pierre Escoubas Nicolas Gilles Edwin De Pauw

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Shedding new lights on the recombinant β-KTx neurotoxin from <em>Tityus serrulatus</em>: heterologous expression, structural and functional characterization.

Fernanda Gobbi Amorim Johara Boldrini-França Francielle Almeida Cordeiro Felipe Augusto Cerni Manuela Berto Pucca

Neurotoxins are the major responsible for the symptoms caused by Tityus serrulatus envenoming due to their actions on ion-channels of excitable cells. However, the structural and functional analyses of these toxins is difficult due to the low amount of purified toxin obtained from the crude venom. The combination of "-omics" techniques allows the precise identification of novel components with biotechnological applications enabling its heterologous expression. We reported the heterologous expression of the recombinant Ts19 (rTs19), a β-KTx neurotoxin, and their structural and functional characterizations. The cDNA encoding rTs19 was obtained from Tityus serrulatus venom gland transcriptome, cloned into pPICZαA plasmid and transformed into cells of KM71H Pichia pastoris strain. rTs19 was purified by immobilized metal affinity and C18 chromatography procedures and showed a higher expression after 96h of induction in buffered methanol-complex medium at 30°C. The expression of the toxin was confirmed by western blot using anti-His-tag antibody. In addition, rTs19 showed a molar mass of 6555.05 Da confirmed by FT-ICR high-resolution mass spectrometry (Solarix, Bruker). After reduction and alkylation, MALDI-TOF analyses (Ultraflex II, Bruker) confirmed the three disulfide bridges of the toxin. rTs19 was sequenced by enzymatic digestion using trypsin and MS/MS fragmentation in a Q-TOF mass spectrometer (SynaptG2, Waters). Electrophysiological experiments and voltage-clamp with two microelectrodes on Xenopus laevis oocytes were performed to screen the action of rTs19 over 16 different subtypes of Kv channels. The rTs19 interacts with potassium channels, blocking Kv1.4 and hERG channels with a high potency. These results demonstrated the first recombinant expression of a β-KTx neurotoxin from Tityus serrulatus. P. pastoris expression system seems to be an efficient, rapid and cheap method for obtaining such toxins in a recombinant methodology. Furthermore, these results may open new perspectives of bioprospection of the biological actions of rTs19.

Heterologous expression

Neurotoxin

Heterologous

10.3390/iect2021-09132

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

Loïc Quinton Julien Echterbille Nicolas Gilles Edwin De Pauw

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Ion Mobility Mass Spectrometry as a Potential Tool To Assign Disulfide Bonds Arrangements in Peptides with Multiple Disulfide Bridges

Analytical Chemistry (2013)

Julien Echterbille Loïc Quinton Nicolas Gilles Edwin De Pauw

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 tandem mass spectrometry (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 nonclassical ions. MS/MS alone does not allow either the cysteine pairing or 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 bridge 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. Cysteine connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference.

Ion-mobility spectrometry

Disulfide Linkage

Collision-induced dissociation

Sequence (biology)

10.1021/ac303686w

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Citations (26)

Mass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors

Analytical and Bioanalytical Chemistry (2015)

Camila Takeno Cologna Nicolas Gilles Julien Echterbille Michel Degueldre Denis Servent

10.1007/s00216-015-8692-4

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