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See detailGender differences in responses in Gammarus pulex exposed to BDE-47: a gel-free proteomic approach
Gismondi, Eric ULg; Mazzucchelli, Gabriel ULg; De Pauw, Edwin ULg et al

in Ecotoxicology & Environmental Safety (2015), 122

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and ... [more ▼]

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and females under control conditions (unexposed) and after 96 h exposure to BDE-47. Using gel-free proteomic analysis, we have identified 45 proteins, of which 25 were significantly differently expressed according to sex and/or BDE-47 exposure. These proteins were involved in several biological processes such as energy metabolism, chaperone proteins, or transcription/translation. In unexposed amphipods, 11 proteins were significantly over-expressed in females, and 6 proteins were over-expressed in males. Under BDE-47 stress, 7 proteins were differently impacted according to sex. For example, catalase was over-expressed in exposed females and under-expressed in exposed males, as compared to respective controls. Conversely, proteins involved in energy metabolism were up-regulated in males and down-regulated in females. Our proteomic study showed differences in responses of males and females to BDE-47 exposure, emphasizing that sex is a confounding factor in ecotoxicological assessment. However, due to the limited information existing in databases on Gammarids, it was difficult to define a BDE-47 mechanism of action. The gel-free proteomic seems to be a promising method to develop in future ecotoxicological studies and thus, to improve our understanding of the mechanism of action of xenobiotics. [less ▲]

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See detailImpact of Regiella insecticola infection on EBF production in Acyrthosiphon pisum Harris
Bauwens, Julien ULg; Mazzucchelli, Gabriel ULg; De Pauw, Edwin ULg et al

Conference (2015, November 06)

Symbionts of aphids influence their host from many points of view. We investigate the potential influence of bacterial symbionts on the production and emission of the aphid alarm pheromone, E-β-franesene ... [more ▼]

Symbionts of aphids influence their host from many points of view. We investigate the potential influence of bacterial symbionts on the production and emission of the aphid alarm pheromone, E-β-franesene. Some trends could be observed in the total EβF production. Particularly, aphid strains infected by Buchnera only seemed to produce less alarm pheromone. By contrast, the presence of Regiella insecticola seemed to increase EβF production. Mevalonate pathway was investigated by RT-qPCR. This analysis showed a slightly lower transcription level o mIPPS in Regiella-infected strains. This enzyme is involved in the last step of EβF production. By contrast, two enzymes involved respectively in the linkage and release of farnesyl moeities on proteins c-terminal ends. Escape tests were conducted to assay if these results were traduced by differential behavior in front of a predator. Preliminary results showed significantly higher dropping behavior for Regiella-infected strains. [less ▲]

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See detailSTUDY OF FURAN FORMATION DURING COFFEE BREWING
Alsafra, Zouheir ULg; De Pauw, Edwin ULg; Eppe, Gauthier ULg et al

Poster (2015, November 05)

Furan (C4H4O) is a small cyclic ether, classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to human (group 2B) [1,2]. It has been found in many foodstuffs ... [more ▼]

Furan (C4H4O) is a small cyclic ether, classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to human (group 2B) [1,2]. It has been found in many foodstuffs processed by heat treatments [3], where it is formed through multiple pathways, such as Maillard reaction, carbohydrates degradation or lipid oxidation [4,5]. A very popular beverage that is also known as the most contaminated foodstuff by furan is coffee. The high contamination level is known to be related to the bean roasting process occurring at high temperature in anaerobic conditions. Macrae and coworkers in 1985, showed that a small amount of furan precursors remain in coffee even after the roasting and grounding process. The aim of this work is to study the possibility of furan formation in coffee beverages and related cross-products from the remaining traces of precursors within the brewing process conditions. [less ▲]

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See detailHigh-throughput sequencing of toxins with pharmacological interest: proof of concept and first applications
Echterbille, Julien ULg; Degueldre, Michel ULg; Boulanger, Madeleine ULg et al

Conference (2015, September 28)

Animal venoms are complex chemical cocktails, comprising wide ranges of biologically active reticulated peptides that target with high selectivity and efficacy varieties of membrane receptors. Assuming ... [more ▼]

Animal venoms are complex chemical cocktails, comprising wide ranges of biologically active reticulated peptides that target with high selectivity and efficacy varieties of membrane receptors. Assuming the fact that each of the 170,000 venomous species reported can produce more than 250 bioactive toxins, at least 40,000,000 bioactive peptides and proteins may be discovered. Among the four described species of mambas, Eastern Jameson’s mamba (Dendroaspis jamesonii kaimosae) venom is the less characterized since only 9 peptides are referenced in database. This work aims at developing a new strategy devoted to the deep analysis of animal venoms. Our approach consists in a first separation of the venom using cation exchange chromatography. Each primary fraction is then purified a second time by classical RP-HPLC. A total of 328 fractions, containing amongst 1 and 4 toxins, are finally collected. MALDI-MS analysis of each fraction is done in order (1) to obtain information about masses and (2) to obtain sequences of toxins thanks to MALDI-In Source Decay (ISD) dissociation coupled with on MALDI target plate reduction of the peptides. ISD has already been demonstrated efficient for toxin sequencing1, and especially when using 1,5-DAN as reducing matrix2. ISD yields to sequences that cover more than 50% of peptide sequences by series of singly charged c-type ions. Thanks to this methodology, we were able to obtain 85% of satisfactory results i.e. spectra giving quite long tags of amino acids (up to 20 residues). As a way to validate our method, a tag coming from ISD spectrum interpretation has found a match in database for an Eastern Jameson’s mamba toxin. The global sequence has then been obtained by extrapolation on the ISD spectrum. Since ISD spectra are simpler than classical MS/MS spectra, automation of spectra interpretation, difficult with other fragmentation techniques (CID, ETD…), is implementable. In the near future, sequences obtained with this approach will be used to direct tests of biological activity through sequence homologies with already known ligands for different kinds of membrane receptors. [less ▲]

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See detailProbing the conformational changes during desolvation of ions using orthogonal mobility methods (CE-IMS)
Far, Johann ULg; Kune, Christopher ULg; Delvaux, Cédric ULg et al

Poster (2015, July 29)

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium ... [more ▼]

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium conformation in the gas phase. The pathway to reach the thermodynamically stable conformation involves crossing potential barriers of different heights. When these barriers are too high compared to the internal energy of the ions, it will result in “partial memories” (as structural preservation) of the conformation in solution. In order to evaluate the effect of the solvent evaporation and of the various collision processes encountered by the ions in the mass spectrometer, we developed two strategies: The first strategy consists in comparing in a single experiment the shape of the ions in solution and in the gas phase. Data are obtained by coupling capillary electrophoresis with Ion Mobility Mass Spectrometry. Drift times in solution and in the gas phase are directly compared. Deviations from their correlation points out changes in folding upon desolvatation. Preliminary results show that among peptides issued from tryptic digest of BSA some of them clearly change their conformation during desolvatation. The second strategy consists in probing changes of conformation once the ions are in the gas phase. The ions are rapidly heating by collisions ions during their transfer to the IMS. The heating is obtained by increasing their collision energy, rapidly followed by thermalisation in the IMS cell. The ions may be kinetically trapped in their new conformations. This allows comparing barriers between different ions geometries. In summary this work intends to evaluate the extent of conformational “memory” of the ions of different nature for best experimental condition allowing “native mass spectrometry” [less ▲]

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See detailCONTRIBUTION OF ION MOBILITY FOR STRUCTURAL ANALYSIS AND ANALYTICAL CHEMISTRY: THE USE OF PROBE LIGANDS AND SELECTIVE IMS SHIFT REAGENTS
Kune, Christopher ULg; Far, Johann ULg; Delvaux, Cédric ULg et al

Conference (2015, July 28)

Ion mobility is a gas phase separation technique sensitive to the Collisional Cross Section (CCS) difference of ions (as CCS/ΔCCS). It discriminates isobaric and isomeric ions when CCS difference is ... [more ▼]

Ion mobility is a gas phase separation technique sensitive to the Collisional Cross Section (CCS) difference of ions (as CCS/ΔCCS). It discriminates isobaric and isomeric ions when CCS difference is larger than the instrumental resolution (roughly 50). To overcome the usual resolution of ion mobility (IM), it is necessary to use new strategies in addition to the optimization of the ion mobility parameters. This work proposes a new method to bypass this limitation while providing additional structural information by the use of Selective Shift Reagents (SSR). A SSR can specifically bind with a target ion depending of their physicochemical properties like chemical groups, steric hindrance, polarity, space charge effects… In this strategy, the choice of SSR is fundamental. SSR could be empirically selected or assisted and designed by computational chemistry prediction. SSR can be used as a chemical probe which can support physicochemical properties and help or confirm hypotheses for structural elucidation. They can also drastically change the CCS of a target ion present in a complex mixture (e.g. biological origin sample) as shifting reagent for e.g. quantification purpose. Models used for the proof of concept have been selected in order to lead to an expected or predictive result. Firstly crown ethers have been used as SSR in IMS to shift the protonated valine drift time from the protonated proline drift time according to their chemical groups and especially amino groups. The selectivity of SSR leads to an improved separation between valine and proline. Secondly three structural isomers of diaminonaphthalene were investigated experimentally and theoretically using computational chemistry support after the addition of different crown ethers or β-cyclodextrin as SSR to improve the separation of these isomers by IMS. Finally, the concept of SSR was successfully applied to biological origin samples to elucidate structure and allows the quantification of selenium (Se) containing compounds present in an aqueous extract of Se rich yeast. [less ▲]

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See detailTravelling-wave ion mobility time-of-flight mass spectrometry as an alternative strategy for screening of multi-class pesticides in fruits and vegetables
Goscinny, Séverine ULg; Joly, Laure; De Pauw, Edwin ULg et al

in Journal of Chromatography. A (2015), 1405

This paper reports a novel approach to screening multi-class pesticides by ion mobility timeof- flight mass spectrometry detection. Nitrogen was selected as mobility gas. After optimization of the ... [more ▼]

This paper reports a novel approach to screening multi-class pesticides by ion mobility timeof- flight mass spectrometry detection. Nitrogen was selected as mobility gas. After optimization of the different ion mobility parameters, determination of matrix effect on the drift times was conducted using different matrix extracts. The results showed that drift time values are not influenced by the matrix and also are independent of the concentration within the working range for 100 pesticides tested, making drift time a powerful additional identification tool. Based on statistics, 2% variation criteria provides a good fit for all the pesticides targeted, and could be considered as a maximum acceptable criteria associated with the drift time parameter for identification purpose. This 2% value is in agreement with already reported criteria, for instance, for GC or LC retention time in European documents. Finally, the well-known feature of mobility to separate complex mixtures was also tested to obtain purified extracted mass spectra of pesticides in fruit extract. [less ▲]

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See detailEnergetics and Structural Characterization of Isomers Using Ion Mobility and Gas-phase H/D Exchange: Learning from Lasso Peptides
Hanozin, Emeline ULg; Morsa, Denis ULg; De Pauw, Edwin ULg

in Proteomics (2015), early view

State-of-the-art characterization of proteins using mass spectrometry namely relies on fragmentation methods which allows exploring featured dissociative reaction pathways. These pathways are often ... [more ▼]

State-of-the-art characterization of proteins using mass spectrometry namely relies on fragmentation methods which allows exploring featured dissociative reaction pathways. These pathways are often initiated by a series of potentially informative mass-constant conformational changes that are nonetheless frequently overlooked by lack of adequate investigation techniques. In the present study, we propose a methodology to readily address both structural and energetic aspects of stereoisomerization reactions using ion mobility coupled with mass spectrometry. To this end, a commercial spectrometer was used as a reactor comprising an energy resolved collisional activation step intended at promoting controlled conformational changes and a structural assignment step dedicated to the identification of the generated isomers. This identification relies on ion mobility and other on-line coupled techniques, namely an originally designed gas-phase H/D exchange experiment. We here apply this methodology to characterize the isomerization kinetics of capistruin, a 19-residue long lasso-folded peptide. We expect this approach to bring insights into the physical origin of global dissociation thresholds monitored in tandem mass spectrometry experiments and to set a promising basis for quantitative investigations of the stability of different molecular folds. [less ▲]

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See detailDe novo sequencing using MELD proteolysis coupled to a "sequence assembly" algorithm
Mazzucchelli, Gabriel ULg; Zimmerman, Tyler; Smargiasso, Nicolas ULg et al

in 63rd ASMS Conference Proceedings, May 30 - June 4 2015, St. Louis, MO (2015, June)

Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic ... [more ▼]

Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic peptides. Peptide analysis has the advantage to be compatible with well-known workflows. Nevertheless the connectivity between peptides is lost. Taking these considerations into account, a specific digestion method and a sequence assembly software were developed. The MELD method relies on a combination of MultiEnzymatic AND Limited proteolytic Digestions. The MELD generates in a single experiment numerous different peptides with miss-cleavages that overlap when aligned on the matching protein sequence. The two major benefices are an increased probability to obtain the entire protein sequence and a redundancy in the protein sequence matches. Methods The MELD consists in two parallel 2h digestions both using an optimized protease mixture. The mixtures are composed of the same proteases but in different relative quantities. Analyses were performed by UPLC-Orbitrap (IClass, Waters, QExactive, Thermo). Data were processed with PEAKS (BSI) to generate de novo sequence candidates. After data importation into our software, a seed sequence is set or can be found automatically. The software extends the seed sequence in both directions with moving windows of three amino acids, plus a fourth one to be added. The added amino acid is validated in several ways, including by the total frequency of occurrence, by larger windows of aa, by the local spectrum-derived confidence, and by combinations of these factors. Preliminary Data The MELD protocol was first validated by applying a traditional database search workflow on several commercial proteins with an inter-day and inter-individual procedure. These experiments showed 100% sequence coverage for each protein analyzed, involving information on peptide identity and modifications localization. Strong confident identification was obtained due to multiple overlapping peptides matches with the given sequence and with a high number of overlapping peptides assignments. The analysis of the four proteins provided the following results: HSA, Myoglobin and Lysozyme were identified with 100% sequence coverage with respectively 890, 300 and 120 unique peptides (CV<10%, peptide FDR<0.1%). The variable region of each heavy and light chains of Adalimumab antibody were identified with 100% sequence coverage. With the MELD, an average of 10 different peptides covering each sequence stretch of the protein could be obtained. The combinatory effect of the multiple enzymes used and the limited digestions leads to an increased robustness, very high confidence identifications and allows clear localization of PTMs. The MELD protocol as presented here and tested on several pure proteins digested in solution, certainly improves the general "bottom-up" strategy applied for highly confident protein identification and would allow better protein characterization, even for those having PTMs. In addition, in our analysis, each fragment position of the entire protein sequence was evidenced either by a "y" or a "b" fragment ion. This high and confident amount of information enables extensive de novo sequencing using PEAKS software, followed by application of our “sequence assembly” algorithm. The first version of our assembling tool on MELD experimental data generated long sequence tags, up to 90 amino acids long. [less ▲]

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See detailMultiple analyses of microbial communities applied to the gut of the wood-feeding termite Reticulitermes flavipes fed on artificial diets
Tarayre, Cédric ULg; Bauwens, Julien ULg; Mattéotti, Christel et al

in Symbiosis (2015)

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on ... [more ▼]

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on poplar wood (original diet) and artificial diets consisting of crystalline cellulose (with and without lignin), α-cellulose (with and without lignin) and xylan. The termites were then dissected and the protist communities were analyzed through microscopy, leading to the conclusion that protist species are strongly influenced by diets. BIOLOG ECO Microplates® were used to assess the metabolic properties of the different types of consortia, highlighting strong differences on the basis of principal component analysis and calculation of similarity rates. The microorganisms were cultivated in liquid media corresponding to the artificial diets before being characterized through a metagenetic analysis of gut microbiota (16S ribosomal DNA). This analysis identified several phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Nitrospirae, OP9, Planctomycetes, Proteobacteria, Spirochaetes, TM6, Tenericutes, Verrucomicrobia and WS3. The OTUs were also determined and confirmed the abundance of Proteobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia. It was possible to isolate several strains from the liquid media, and one bacterium and several fungi were found to produce interesting enzymatic activities. The bacterium Chryseobacterium sp. XAvLW produced α-amylase, β-glucosidase, endo-1,4-β-D-glucanase, endo-1,4-β-D-xylanase and filter paper-cellulase, while the fungi Sarocladium kiliense CTGxxyl and Trichoderma virens CTGxAviL generated the same activities added with endo-1,3-β-D-glucanase. [less ▲]

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See detailStudy of N-linked glycosylation in different life stages of the red flour beetle (Tribolium castaneum)
Smargiasso, Nicolas ULg; Walski, Tomasz; Van Damme, Els et al

Poster (2015, June)

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