Publications of Loïc Quinton
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See detailInactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans
Herman, Xavier; Far, Johann ULiege; Courtoy, Adeline et al

in Frontiers in Plant Science (2021)

Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced ... [more ▼]

Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms. [less ▲]

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See detailVenomics approach reveals a high proportion of Lactrodectus-like toxins in Steatoda nobilis venom - First link to post-bite symptomology
Dunbar, John; Fort, Antoine; Redureau, Damien ULiege et al

Poster (2020, September)

The Noble false widow spider Steatoda nobilis has expanded its range globally and may represent a potential threat to native ecosystems and public health. Envenomations can result in local and systemic ... [more ▼]

The Noble false widow spider Steatoda nobilis has expanded its range globally and may represent a potential threat to native ecosystems and public health. Envenomations can result in local and systemic neurotoxic symptoms, similar to true black widows (genus Latrodectus). We used transcriptomic and proteomic cutting-edge approaches to deeply characterise S. nobilis venom. Among the toxins, the most represented in numbers are α-latrotoxins, 𝛿-latroinsectotoxins and latrodectins, which were first characterised from black widow venoms. Approximately two-thirds of the venom is composed of Latrodectus-like toxins. We present symptomology from 23 cases (15 unpublished) of S.nobilis envenomations confirming necrosis and Latrodectus-like symptoms such as debilitating pain, tremors, fatigue, nausea and hypotension. The continued rising numbers of S. nobilis will undoubtedly result in further bites and this study will help provide the medical community with a better understanding of the potential medical outcomes from bites by this species and alert them to the possibility of medically important outcomes. [less ▲]

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See detailEffect of ion counts stability during dynamically harmonized FT-ICR-MSI
Tiquet, Mathieu ULiege; La Rocca, Raphaël ULiege; Far, Johann ULiege et al

Poster (2019, April 01)

Mass spectrometry imaging of complex biological samples often require high spectral resolution and mass accuracy to properly distinguish all isobaric compounds. To achieve such requirement dynamically ... [more ▼]

Mass spectrometry imaging of complex biological samples often require high spectral resolution and mass accuracy to properly distinguish all isobaric compounds. To achieve such requirement dynamically harmonized FT-ICR analysers offer best results. However, mass accuracy below expectation due to masses shifting between pixels observed when performing image acquisitions. In this work we show a link between the mass shifting phenomenon andd the large variations observed in total ion current during image acquisitions. This work propose solve the mass shift by optimisation in sample preparation and acquisition parameters to stabilise the fluctuation of the total ion current in FT-ICR-MSI. [less ▲]

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See detailProteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins
Amorim, F. G.; Longhim, H. T.; Cologna, C. T. et al

in Journal of Venomous Animals and Toxins Including Tropical Diseases (2019), 25

Background: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and ... [more ▼]

Background: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom's fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology. © The Author(s). [less ▲]

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See detailAnalytical tool for lipopeptide identification
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; Far, Johann ULiege et al

Conference (2018, November 30)

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See detailCombination of Capillary Electrophoresis and Ion mobility coupled to Mass Spectrometry and Theoretical Calculations for cysteine connectivity identification in peptides bearing two intra-molecular disulfide bonds
Delvaux, Cédric ULiege; Massonnet, Philippe ULiege; Kune, Christopher ULiege et al

Conference (2018, November 06)

Disulfide bonds are post translational modification playing essential roles in the biological activity and stability of numerous peptides and proteins. Intra-molecular disulfide bonds are found in various ... [more ▼]

Disulfide bonds are post translational modification playing essential roles in the biological activity and stability of numerous peptides and proteins. Intra-molecular disulfide bonds are found in various natural-occurring peptides such as animal venoms. In such peptides, the appropriate cysteine connectivity provides the required conformation for efficient binding to their molecular targets, which ensures their bioactivity. The characterization of cysteine pairing is still a challenging issue in the analysis of peptides targeting pharmaceutical or pharmacological utilizations. Thus, the use of sensitive, robust and efficient characterization techniques to access the cysteine pairing is crucial. In our workflow,the separation of the disulfide isomers of three peptides bearing two intra-molecular disulfide bonds but different cysteine connectivities have been tested using Capillary Zone Electrophoresis (CZE) and Ion Mobility (IM) coupled to Mass Spectrometry (MS). Results show that CZE-MS and IM-MS act as complementary techniques to unambiguously determine the cysteine connectivity of a given peptide. Indeed, the combination of the relative migration time to a reference peptide in CZE-MS, the drift time in IM-MS and the generation of fragments by Collision Induced Dissociation (CID) led to the attribution of the disulfide connectivities in all studied cases. Finally, theoretical calculations were performed to model the different structures in gas phase and solution, supporting the experimental observations on the basis of the predicted physicochemical properties. [less ▲]

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See detailExploitation de la plateforme e-Campus pour l’apprentissage de la Chimie de Première année pour les étudiants non acquis à la matière
Kune, Christopher ULiege; Quinton, Loïc ULiege

Scientific conference (2017, November 23)

De nos jours, les étudiants sont de plus en plus ouverts à l’e-Learning. La plateforme e-Campus, de par sa facilité à rendre disponible des contenus et des activés en ligne, permet à tout corps enseignant ... [more ▼]

De nos jours, les étudiants sont de plus en plus ouverts à l’e-Learning. La plateforme e-Campus, de par sa facilité à rendre disponible des contenus et des activés en ligne, permet à tout corps enseignant de répondre à cette demande grandissante. Pour les étudiants en première année de kinésithérapie et sciences de la motricité, l’e-Learning est devenu un complément incontournable du cours de Chimie. Lors de cette présentation, nous aborderons le développement de ce projet E-Learning pour le cours de chimie, de l’idée à la réalisation, en découvrant les outils offerts par la plateforme E-Campus, tels les questionnaires en ligne, les forums, les médiathèques… [less ▲]

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See detailIn-depth glyco-peptidomics approach reveals unexpected diversity of glycosylated peptides and atypical post-translational modifications in dendroaspis angusticeps snake venom
Degueldre, Michel ULiege; Echterbille, Julien; Smargiasso, Nicolas ULiege et al

in International Journal of Molecular Sciences (2017), 18(11), 2483

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the ... [more ▼]

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. [less ▲]

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See detailAPETx4, a novel sea anemone toxin and a modulator of the cancer-relevant potassium channel KV10.1
Moreels, L.; Peigneur, S.; Galan, D. T. et al

in Marine Drugs (2017), 15(9), 287

The human ether-à-go-go channel (hEag1 or KV10.1) is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit ... [more ▼]

The human ether-à-go-go channel (hEag1 or KV10.1) is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit this channel can be lead compounds in the search for new anticancer drugs. Here, we report the activity-guided purification and electrophysiological characterization of a novel KV10.1 inhibitor from the sea anemone Anthopleura elegantissima. Purified sea anemone fractions were screened for inhibitory activity on KV10.1 by measuring whole-cell currents as expressed in Xenopus laevis oocytes using the two-microelectrode voltage clamp technique. Fractions that showed activity on Kv10.1 were further purified by RP-HPLC. The amino acid sequence of the peptide was determined by a combination of MALDI- LIFT-TOF/TOF MS/MS and CID-ESI-FT-ICR MS/MS and showed a high similarity with APETx1 and APETx3 and was therefore named APETx4. Subsequently, the peptide was electrophysiologically characterized on KV10.1. The selectivity of the toxin was investigated on an array of voltage-gated ion channels, including the cardiac human ether-à-go-go-related gene potassium channel (hERG or Kv11.1). The toxin inhibits KV10.1 with an IC50 value of 1.1 µM. In the presence of a similar toxin concentration, a shift of the activation curve towards more positive potentials was observed. Similar to the effect of the gating modifier toxin APETx1 on hERG, the inhibition of Kv10.1 by the isolated toxin is reduced at more positive voltages and the peptide seems to keep the channel in a closed state. Although the peptide also induces inhibitory effects on other KV and NaV channels, it exhibits no significant effect on hERG. Moreover, APETx4 induces a concentration-dependent cytotoxic and proapoptotic effect in various cancerous and noncancerous cell lines. This newly identified KV10.1 inhibitor can be used as a tool to further characterize the oncogenic channel KV10.1 or as a scaffold for the design and synthesis of more potent and safer anticancer drugs. © 2017 by the authors. Licensee MDPI. [less ▲]

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See detailMALDI In-Source Decay, from sequencing to imaging
Debois, Delphine ULiege; Smargiasso, Nicolas ULiege; Demeure, Kevin ULiege et al

in Topics in Current Chemistry (2013), 331

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions ... [more ▼]

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS/MS can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications studies, and finally review MALDI-ISD tissue imaging applications. [less ▲]

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See detailThe usefulness of Ion Mobility-Mass Spectrometry for Small Molecules Analysis
Far, Johann ULiege; Goscinny, Séverine ULiege; Joly, Laure et al

Conference (2012, March)

Detailed reference viewed: 203 (31 ULiège)