Publications of Loïc Quinton
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See detailRapid visualization of lipopeptides and potential bioactive groups of compounds by combining ion mobility and MALDI imaging mass spectrometry
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; La Rocca, Raphaël ULiege et al

in Drug Discovery Today. Technologies (2021)

Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new ... [more ▼]

Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new bioactive compounds and for a better understanding of bacterial antibiotic resistance mechanisms. Coupled with separation techniques such as ion mobility mass spectrometry (IM-MS), the identification of metabolites directly on the image is now possible and does not require additional analysis such as HPLC-MS/MS. In this article, we propose to apply a semi-targeted workflow for rapid IM-MSI data analysis focused on the search for bioactive compounds. First, chemically-related compounds showing a repetitive mass unit (i.e. lipids and lipopeptides) were targeted based on the Kendrick mass defect analysis. The detected groups of potentially bioactive compounds were then confirmed by fitting their measured ion moibilites to their measured m/z values. Using both their m/z and ion mobility values, the selected groups of compounds were identified using the available databases and finally their distribution was observed on the image. Using this workflow on a co-culture of bacteria, we were able to detect and localize bioactive compounds involved in the microbial interaction [less ▲]

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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 detailUse of Capillary Zone Electrophoresis Coupled to Electrospray Mass Spectrometry for the Detection and Absolute Quantitation of Peptidoglycan-Derived Peptides in Bacterial Cytoplasmic Extracts
Delvaux, Cédric ULiege; Dauvin, Marjorie ULiege; Boulanger, Madeleine ULiege et al

in Analytical Chemistry (2021), 93(4), 2342-2350

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and ... [more ▼]

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE–MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE–MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20% [less ▲]

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See detailMass shift in Mass Spectrometry Imaging: comprehensive analysis and practical corrective workflow
Mc Cann, Andréa ULiege; Rappe, Sophie ULiege; La Rocca, Raphaël ULiege et al

in Analytical and Bioanalytical Chemistry (2021)

tentative identification of compounds based on their m/z value. In typical MSI, a spectrum is taken at incremental 2D coordinates (pixels) across a sample surface. Single pixel mass spectra show the ... [more ▼]

tentative identification of compounds based on their m/z value. In typical MSI, a spectrum is taken at incremental 2D coordinates (pixels) across a sample surface. Single pixel mass spectra show the resolving power of the mass analyzer. Mass shift, i.e., variations of the m/z of the same ion(s), may occur from one pixel to another. The superposition of shifted masses from individual pixels peaks apparently degrades the resolution and the mass accuracy in the average spectrum. This leads to low confidence annotations and biased localization in the image. Besides the intrinsic performances of the analyzer, the sample properties (local composition, thickness, matrix deposition) and the calibration method are sources of mass shift. Here, we report a critical analysis and recommendations to mitigate these sources of mass shift. Mass shift 2D distributions were mapped to illustrate its effect and explore systematically its origin. Adapting the sample preparation, carefully selecting the data acquisition settings, and wisely applying post-processing methods (i.e., m/z realignment or individual m/z recalibration pixel by pixel) are key factors to lower the mass shift and to improve image quality and annotations. A recommended workflow, resulting from a comprehensive analysis, was successfully applied to several complex samples acquired on both MALDI ToF and MALDI FT-ICR instruments. [less ▲]

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See detailAdaptive Pixel Mass Recalibration for Mass Spectrometry Imaging Based on Locally Endogenous Biological Signals.
La Rocca, Raphaël ULiege; Kune, Christopher ULiege; Tiquet, Mathieu ULiege et al

in Analytical Chemistry (2021)

Mass spectrometry imaging (MSI) is a powerful and convenient method for revealing the spatial chemical composition of different biological samples. Molecular annotation of the detected signals is only ... [more ▼]

Mass spectrometry imaging (MSI) is a powerful and convenient method for revealing the spatial chemical composition of different biological samples. Molecular annotation of the detected signals is only possible if a high mass accuracy is maintained over the entire image and the m/z range. However, the change in the number of ions from pixel-to-pixel of the biological samples could lead to small fluctuations in the detected m/z-values, called mass shift. The use of internal calibration is known to offer the best solution to avoid, or at least to reduce, mass shifts. Their “a priori” selection for a global MSI acquisition is prone to false positive detection and therefore to poor recalibration. To fill this gap, this work describes an algorithm that recalibrates each spectrum individually by estimating its mass shift with the help of a list of pixel-specific internal calibrating ions, automatically generated in a data-adaptive manner (https://github.com/LaRoccaRaphael/MSI_recalibration). Through a practical example, we applied the methodology to a zebrafish whole-body section acquired at a high mass resolution to demonstrate the impact of mass shift on data analysis and the capability of our algorithm to recalibrate MSI data. In addition, we illustrate the broad applicability of the method by recalibrating 31 different public MSI data sets from METASPACE from various samples and types of MSI and show that our recalibration significantly increases the numbers of METASPACE annotations (gaining from 20 up to 400 additional annotations), particularly the high-confidence annotations with a low false discovery rate. [less ▲]

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See detailStructural interactions define assembly adapter function of a type II secretion system pseudopilin.
Escobar, Cristian A.; Douzi, Badreddine; Ball, Geneviève et al

in Structure (London, England : 1993) (2021)

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The type II secretion system (T2SS), a virulence pathway in many pathogens, belongs to this superfamily ... [more ▼]

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The type II secretion system (T2SS), a virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in understanding of the T2SS is the molecular role of a small "pseudopilin" protein. Using multiple biophysical techniques, we have deciphered how this missing component of the Xcp T2SS architecture is structurally integrated, and thereby unlocked its function. We demonstrate that low-abundance XcpH is the adapter that bridges a trimeric initiating tip complex, XcpIJK, with a periplasmic filament of XcpG subunits. Each pseudopilin protein caps an XcpG protofilament in an overall pseudopilus compatible with dimensions of the periplasm and the outer membrane-spanning secretin through which substrates pass. Unexpectedly, to fulfill its adapter function, the XcpH N-terminal helix must be unwound, a property shared with XcpG subunits. We provide an experimentally validated three-dimensional structural model of a complete type IV filament. [less ▲]

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See detailAn evaluation of the self-assembly enhancing properties of cell-derived hexameric amyloid-β.
Vadukul, Devkee M.; Vrancx, Céline; Burguet, Pierre ULiege et al

in Scientific Reports (2021), 11(1), 11570

A key hallmark of Alzheimer's disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage ... [more ▼]

A key hallmark of Alzheimer's disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein, the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its assembly enhancing properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers. [less ▲]

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See detailUnraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.
Oliveira, Isadora Sousa De; Pucca, Manuela Berto; Wiezel, Gisele Adriano et al

in International journal of biological macromolecules (2021), 178

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully ... [more ▼]

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities. [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 detailA Venomics approach coupled to high-throughput toxin production strategies identifies the first venom-derived melanocortin receptor agonists.
Reynaud, Steve; Ciolek, Justyna; Degueldre, Michel ULiege et al

in Journal of medicinal chemistry (2020)

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most ... [more ▼]

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in E. coli generated a physical bank of 3,597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affect ion channels, the known targets of their toxin scaffolds, but bind to four melanocortin receptors with low micromolar affinities and activate the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists. [less ▲]

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See detailUnravelling chemical mechanisms in microbial interactions by combining thin layer chromatography, ion mobility and MALDI imaging mass spectrometry
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; La Rocca, Raphaël ULiege et al

Conference (2020, June 01)

Mass spectrometry (MS) is a method of choice in microbiology for untargeted detection and identification of bioactive compounds. Mass Spectrometry Imaging (MSI) has led to a growing interest in the in ... [more ▼]

Mass spectrometry (MS) is a method of choice in microbiology for untargeted detection and identification of bioactive compounds. Mass Spectrometry Imaging (MSI) has led to a growing interest in the in situ study of biomolecules produced when microorganisms interact with each other. However, in situ identification is still time-consuming and challenging due to the large chemical diversity contained in each pixel of the samples, resulting in very complex average MS spectra. Here, we propose to exploit the power of combining Kendrick Mass Defect (KMD) analysis and collision cross section (CCS) values using mobility for the characterization of families of related compounds in MSI. The identification of compounds was supported by rapid thin layer chromatography (TLC) separation coupled to MALDI-MS/MS detection. Bacillus velezensis GA1 and Pseudomonas sp. CMR12a were inoculated at different distances (0.5, 1 and 2 cm) on a semi-solid agar-based medium and incubated at 30°C. Regions of interest were cut directly from the Petri dish and transferred to the target ITO plate. This assembly was placed in a vacuum desiccator until completely dry and covered with HCCA matrix (Sunchrom sprayer). Ion mobility in imaging mode was performed using the timsTOF fleX (Bruker Daltonics, Bremen, Germany). TLC separation of ROI extracts is analyzed by MALDI-MS/MS imaging on the rapifleX instrument (Bruker Daltonics) for rapid screening of compounds and on the solariX instrument (Bruker Daltonics) for exact mass and isotopic distribution. The data were processed and integrated with in-house software. The coupling of MALDI-MSI with ion mobility separation brings additional structural features/information. It allows data to be filtered according to a range of CCS values and it improves the confidence level for the identification of detected analytes, in addition to exact mass determination. A relationship between CCS and mass was also performed. In combination with KMD analysis, various families of compounds, such as lipids or lipopeptides could be automatically detected and identified. Through this workflow, the comparison of the three different culture conditions was greatly simplified and highlighted the changes that occur in the metabolism of the bacteria. In particular, we were able to observe a variation within the lipid composition of Pseudomonas sp. CMR12a as a function of distance from the Bacillus velezensis GA1 colony. Finally, TLC separation was successfully optimized for lipopeptides and lipids and validated the identification of detected compounds by simplifying the spectra and allowing image analysis. TLC also enables high throughput, in part due to the parallel imaging of up to 6 traces on a single TLC run. TLC plate matrix coating strategies were compared to optimize the MALDI MS signal. This workflow will also be applied to time-lapse (or time-dependent) experiments, to monitor the bioactive compounds production and migration as a function of the co-culture interaction time. [less ▲]

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See detailCan IM-MS Collision Cross Sections of Biomolecules Be Rationalized Using Collision Cross-Section Trends of Polydisperse Synthetic Homopolymers?
Haler, Jean ULiege; Massonnet, Philippe ULiege; Far, Johann ULiege et al

in Journal of the American Society for Mass Spectrometry (2020), 31(4), 990-995

In the past, we developed a method inferring physicochemical properties from ion mobility mass spectrometry (IM-MS) data from polydisperse synthetic homopolymers. We extend here the method to biomolecules ... [more ▼]

In the past, we developed a method inferring physicochemical properties from ion mobility mass spectrometry (IM-MS) data from polydisperse synthetic homopolymers. We extend here the method to biomolecules that are generally monodisperse. Similarities in the IM-MS behavior were illustrated on proteins and peptides. This allows one to identify ionic species for which intramolecular interactions lead to specific structures. [less ▲]

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See detailData treatment for mass spectrometry
La Rocca, Raphaël ULiege; Quinton, Loïc ULiege; De Pauw, Edwin ULiege

Conference (2020, January 28)

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See detailData processing and interpretation of mass spectrometry imaging using high resolving mass power
Mc Cann, Andréa ULiege; La Rocca, Raphaël ULiege; Kune, Christopher ULiege et al

Poster (2020, January 28)

1 Introduction Mass Spectrometry Imaging (MSI) is a powerful analytical tool allowing untargeted investigation of spatial distribution of molecular species in a large variety of samples, by recording 2D ... [more ▼]

1 Introduction Mass Spectrometry Imaging (MSI) is a powerful analytical tool allowing untargeted investigation of spatial distribution of molecular species in a large variety of samples, by recording 2D distribution of all the detectable compounds in the sample. Over the years, the application of MSI has become increasingly diverse, from bacteria-bacteria interaction understanding to biomarker discovery. To this end, it is necessary to combine high spectral resolution with efficient data processing, in order to maximise the extraction of the information contained in large datasets. Moreover, in addition to deal with thousands of features, pixel-to-pixel mass shift must be taken into account before applying any data-mining tool. For that reason, we propose the combination of a post-acquisition label-free recalibration method and an algorithm to cluster features based on their Kendrick mass defect (KMD) in MSI. 2 Theory KMD consists in a change of basis from the IUPAC mass scale to a Kendrick mass based on the nominal mass of a defined Kendrick mass unit, such as CH2 (here, nominal mass is set at 14 a.u.)1. The KMD is obtained by subtracting the nominal Kendrick mass to the exact Kendrick mass. This mathematical transformation enables to map the detected molecules in mass spectrometry based on their chemical composition. 3 Material and methods MALDIFT-ICR-MS (SolariX XR 9.4T) images were first converted into imzML open format. Each spectrum from the imzML file obtained for an MSI are individually peak picked. Then, our algorithm creates, for each pixel, a linear model linking m/z error and m/z. The model is creating by finding database hit with similar mass shift. Finally, a new imzML file is created by recalibrating each pixel with their estimated linear model. Then, we developed a software to filter features (m/z peaks) based on their KMD from an imzML file. This software calculates first a KMD for each m/z values detected in the mean mass spectrum of the image. The MSI data is then filtered based on KMD value to conserve only ions whose KMD is included in the user-defined KMD range. The reduced ion lists were then divided into different compounds families, based on the repetition of the KMD. Finally, an image is generated for each compounds family. Thereby reducing the number of features of an image2. 4 Results and discussion The KMD filtering method applied on a mouse brain tissue analysis by MSI appears to be biologically relevant since it enabled to map in a single step all members of important families of biomolecules. Among the detected families of biomolecules, lipids shows different distributions and localisations. Some of these lipids belonged to the glycerophosphocholines (GPCs), the hexosylceramides (HexCers), lysophoshocholins (LPCs) and the sphingomyelins (SMs) class. Moreover, The KMD analysis highlighted that some of the detected GPCs on the brain tissue sections from mouse were differentially co-localized, depending on their unsaturation degree. All these results suggested that the KMD could be considered instead of the mass-to-charge (m/z) values classically used for the analysis of images by MSI. The use of our in-house developed software for KMD analysis enables an automated, faster and efficient data analysis of MSI images. 5 Conclusion The combination of recalibration before using Kendrick mass defect mass filter is an essential step to be able to interpret the image reconstruction of chemically-related compounds. This methods speed up the identification process and facilitates the data analysis without losing data information. [less ▲]

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See detailEffective Temperature and Structural Rearrangement in Trapped Ion Mobility Spectrometry
Morsa, Denis ULiege; Hanozin, Emeline ULiege; Eppe, Gauthier ULiege et al

in Analytical Chemistry (2020)

Modern ion mobility instrumentations are typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during the analysis. Here, we ... [more ▼]

Modern ion mobility instrumentations are typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during the analysis. Here, we quantitatively assessed the internal heating experienced by ions during trapped ion mobility spectrometry (TIMS) experiments. To this end, the fragmentation yields of fragile benzylpyridinium “thermometer” ions were monitored during both the accumulation and analysis steps inside the TIMS tunnel. The corresponding fragmentation rate constants were translated into a vibrational effective temperature Teff,vib. Our results demonstrate significant fragmentation upstream and inside the TIMS tunnel that corresponds to Teff,vib ≈ 510 K during both the accumulation and analysis steps. Broadening our scope to cytochrome c and lysozyme, we showed that although compact “native” folds can be preserved, the collision cross section distributions are highly sensitive to the transmission voltages and the analysis timescale. Our results are discussed with regard to Teff,vib data previously acquired on traveling-wave (TWIMS) ion mobility in the context of native mass spectrometry and conformational landscape exploration. [less ▲]

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See detailVenomics Approach Reveals a High Proportion of Lactrodectus-Like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis.
Dunbar, John P.; Fort, Antoine; Redureau, Damien et al

in Toxins (2020), 12(6),

The noble false widow spider Steatoda nobilis originates from the Macaronesian archipelago and has expanded its range globally. Outside of its natural range, it may have a negative impact on native ... [more ▼]

The noble false widow spider Steatoda nobilis originates from the Macaronesian archipelago and has expanded its range globally. Outside of its natural range, it may have a negative impact on native wildlife, and in temperate regions it lives in synanthropic environments where it frequently encounters humans, subsequently leading to envenomations. S. nobilis is the only medically significant spider in Ireland and the UK, and envenomations have resulted in local and systemic neurotoxic symptoms similar to true black widows (genus Latrodectus). S. nobilis is a sister group to Latrodectus which possesses the highly potent neurotoxins called α-latrotoxins that can induce neuromuscular paralysis and is responsible for human fatalities. However, and despite this close relationship, the venom composition of S. nobilis has never been investigated. In this context, a combination of transcriptomic and proteomic cutting-edge approaches has been used to deeply characterise S. nobilis venom. Mining of transcriptome data for the peptides identified by proteomics revealed 240 annotated sequences, of which 118 are related to toxins, 37 as enzymes, 43 as proteins involved in various biological functions, and 42 proteins without any identified function to date. Among the toxins, the most represented in numbers are α-latrotoxins (61), δ-latroinsectotoxins (44) and latrodectins (6), all of which were first characterised from black widow venoms. Transcriptomics alone provided a similar representation to proteomics, thus demonstrating that our approach is highly sensitive and accurate. More precisely, a relative quantification approach revealed that latrodectins are the most concentrated toxin (28%), followed by α-latrotoxins (11%), δ-latroinsectotoxins (11%) and α-latrocrustotoxins (11%). Approximately two-thirds of the venom is composed of Latrodectus-like toxins. Such toxins are highly potent towards the nervous system of vertebrates and likely responsible for the array of symptoms occurring after envenomation by black widows and false widows. Thus, caution should be taken in dismissing S. nobilis as harmless. This work paves the way towards a better understanding of the competitiveness of S. nobilis and its potential medical importance. [less ▲]

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See detailVenomics of the asp viper Vipera aspis aspis from France
Giribaldi, J.; Kazandjian, T.; Amorim, F. G. et al

in Journal of Proteomics (2020), 218

The asp viper Vipera aspis aspis is a venomous snake found in France, and despite its medical importance, the complete toxin repertoire produced is unknown. Here, we used a venomics approach to decipher ... [more ▼]

The asp viper Vipera aspis aspis is a venomous snake found in France, and despite its medical importance, the complete toxin repertoire produced is unknown. Here, we used a venomics approach to decipher the composition of its venom. Transcriptomic analysis revealed 80 venom-annotated sequences grouped into 16 gene families. Among the most represented toxins were snake venom metalloproteases (23%), phospholipases A2 (15%), serine proteases (13%), snake venom metalloprotease inhibitors (13%) and C-type lectins (12%). LC-MS of venoms revealed similar profiles regardless of the method of extraction (milking vs defensive bite). Proteomic analysis validated 57 venom-annotated transcriptomic sequences (>70%), including one for each of the 16 families, but also identified 7 sequences not initially annotated as venom proteins, including a serine protease, a disintegrin, a glutaminyl-peptide cyclotransferase, a proactivator polypeptide-like and 3 aminopeptidases. Interestingly, phospholipases A2 were the dominant proteins in the venom, among which included an ammodytoxin B-like sequence, which may explain the reported neurotoxicity following some asp viper envenomations. In total, 87 sequences were retrieved from the Vipera aspis aspis transcriptome and proteome, constituting a valuable resource that will help in understanding the toxinological basis of clinical signs of envenoming and for the mining of useful pharmacological compounds. Biological significance: The asp viper (Vipera aspis aspis) causes several hundred envenomations annually in France, including unusual cases with neurological signs, resulting in one death per year on average. Here, we performed a proteotranscriptomic analysis of V. a. aspis venom in order to provide a better understanding of its venom composition. We found that, as in other Vipera species, phospholipase A2 dominates in the venom, and the presence of a sequence related to ammodytoxin B may explain the reported neurotoxicity following some asp viper envenomations. Thus, this study will help in informing the toxinological basis of clinical signs of envenoming. © 2020 Elsevier B.V. [less ▲]

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See detailA comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms
Barbosa, Luana Gonçalves; Costa, Tassia Rafaela; Borges, Isabela Pacheco et al

in International Journal of Biological Macromolecules (2020)

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two ... [more ▼]

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action. [less ▲]

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See detailPioneering Study on Rhopalurus crassicauda Scorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase.
Abreu, Caio B.; Bordon, Karla C. F.; Cerni, Felipe A. et al

in Frontiers in immunology (2020), 11

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including ... [more ▼]

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Na(v)1.4, Na(v)1.6, and BgNa(v)1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings. [less ▲]

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