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See detailImaging MS: strategies for the identification of analytes
Debois, Delphine ULg; Smargiasso, Nicolas ULg; Jourdan, Emmanuel et al

Scientific conference (2014, April 04)

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See detailNew mass spectrometry based methodology to sequence a whole snake venom
Echterbille, Julien ULg; Boulanger, Madeleine; Degueldre, Michel ULg et al

Poster (2014, February 10)

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 sequencing, and especially when using 1,5-DAN as reducing matrix. 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 detailIsolation of an amylolytic chrysophyte, Poterioochromonas sp. from the digestive tract of the termite R. santonensis
Tarayre, Cédric ULg; Bauwens, Julien ULg; Brasseur, Catherine ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2014), 18(1),

The aim of this work was the isolation and cultivation of amylolytic protists living in the digestive tract of the termite Reticulitermes santonensis (Feytaud). A chrysophyte identified as ... [more ▼]

The aim of this work was the isolation and cultivation of amylolytic protists living in the digestive tract of the termite Reticulitermes santonensis (Feytaud). A chrysophyte identified as Poterioochromonas sp. was isolated in a special medium containing rice grains as a source of carbon and nitrogen. Then, the protist was grown in a medium containing starch as a carbon source, tryptone, and a phosphate buffer at different pH values (5, 6 and 7). Yeast extract was added or not. Ciprofloxacin was used to avoid the bacterial development. Other antibiotics were also tested but showed an inhibitive effect on the growth of Poterioochromonas sp. Yeast extract allowed reaching 1.9 (pH 5), 2.3 (pH 6) and 2.2 (pH 7) times higher final cell concentrations, and 2.8 (pH 5), 2.8 (pH 6) and 2.2 (pH 7) times higher biomass yields. The starch concentration did not decrease in the medium until 3 and 4 days of culture, with and without yeast extract, respectively. Eight days of culture were necessary for hydrolyzing the starch completely, with and without yeast extract. Maltose and maltotriose were detected in the culture media and were hydrolyzed progressively. Maximal maltose concentrations were 0.68, 0.66 and 0.51 g.l-1 in the medium containing yeast extract. Maltotriose concentrations were only 0.17, 0.14 and 0.12 g.l-1. Other glucose oligomers were also detected but in lower quantities. It was determined that the protist developed a weak amylase activity, particularly at a weakly acidic pH (5-6). Such a pH also allowed a better growth of the protist. A maximal amylase activity of 112 nkat.l-1 was measured with yeast extract at pH 5. No other enzymatic activity (protease, cellulase or xylanase) was detected except amylase. The degradation products of starch which were obtained by enzymatic hydrolysis allow the identification of α-amylase, amyloglucosidase and possibly β-amylase activities. [less ▲]

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See detailInnovative analytical strategies for small molecules analysis by ion-mobility mass spectrometry
Eppe, Gauthier ULg; Goscinny, Séverine; Far, Johann ULg et al

Conference (2014, January)

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See detailBioactive Intraocular Lens – A Strategy to Control Secondary Cataract
Huang, Yi-Shiang ULg; Bertrand, Virginie ULg; Bozukova, Dimitriya et al

in IFMBE Proceedings (2014), 41

Cataract is the opacity of the lens, causing impairment of vision or even blindness. Today, a surgery is still the only available treatment. The intraocular lens (IOL) is a polymer implant designed to ... [more ▼]

Cataract is the opacity of the lens, causing impairment of vision or even blindness. Today, a surgery is still the only available treatment. The intraocular lens (IOL) is a polymer implant designed to replace the natural lens in the cataract surgery. However, the bioinert materials could not satisfy the unmet need in the secondary cataract control. Posterior capsular opacification (PCO, or Secondary Cataract), characterized by a thick and cloudy layer of lens epithelial cells (LECs), is the most common postoperative complication. In our research, a bioactive molecule is immobilized onto the conventional acrylic hydrophilic polymer pHEMA (Poly(2-hydroxyethyl methacrylate)) using oxygen plasma treatment followed by deposition. The RGD peptide sequence, being well-known for its ability to promote cellular attachment by binding to integrin receptors, is designed to stimulate the adhesion of LECs on the IOL. Our data show the peptide immobilized biomaterial not only exhibits similar optical property, but also reveals enhanced biological properties in cell adhesion and cell morphology maintenance. By means of surface functionalization of IOL to stimulate LECs adhesion, the secondary cataract could be controlled. [less ▲]

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See detailAdvances in proteomics for the FP7 Venomics project - Disulfide bridge assignement task
Massonnet, Philippe ULg; Upert, Gregory; Pastor, Alexandra et al

Scientific conference (2013, December 18)

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See detailTandem MS of -new- antibiotics from Bacillus guided by MALDI Mass Spectrometry Imaging
Debois, Delphine ULg; Jourdan, Emmanuel; Cawoy, Hélène ULg et al

Conference (2013, December 05)

Generally, an antibiotic is thought to have a role in antagonism simply because the producing strain is known to exhibit a potential for pathogen growth inhibition. Some genetic approaches such as PCR ... [more ▼]

Generally, an antibiotic is thought to have a role in antagonism simply because the producing strain is known to exhibit a potential for pathogen growth inhibition. Some genetic approaches such as PCR using specific primers or genome mining using known sequence data of close relatives are also used. Nevertheless, none of these methods allows stating for a link between a specific compound and the observed antagonism. Yet MALDI Mass Spectrometry Imaging (MSI) is a powerful tool to decipher the chemical messengers exchanged by two protagonists [1,2,3;]. Tandem mass spectrometry (MS/MS) may be also used, either on extracts [2,3] or directly on the microbial colonies [4]. The presentation will thus be focused on two examples of application of MALDI MSI combined to in situ tandem mass spectrometry. The first presented case will be the antagonism between soilborne strain Paenibacillus polymyxa Pp56 and the fungal phytopathogen Fusarium oxysporum. Using MALDI MSI, we were able to precisely localize each detected antibiotic, allowing discriminating which LI-F lipopeptides (fusaricidin) were really active against the pathogen progression. Besides, the use of in situ MS/MS allowed us to sequence the peptide moiety of several LI-F lipopeptides, showing that some of them are actually a mixture of several forms. The second example concerns the metabolites that are released by Bacillus amyloliquefaciens S499 cells following their inoculation on 7 days old tomato roots. We developed specific bioassays for time-course monitoring by MALDI MSI. First analyses revealed an efficient secretion of surfactin by Bacillus cells after 3 days when colonization as biofilm-structured populations is well established. Even if the composition of antibiotic mixture does not greatly evolve over time, after long incubation periods (32 or 35 days post inoculation), new series of compounds are detected in the tomato root -surrounding medium. Structural analysis based on exact mass measurements and MS/MS experiments, performed directly on the semi-solid agar medium, allowed us to identify these compounds as new variants of surfactins. [1] Barger, S., et al., Anton Leeuw Int J G, 2012, 102, 435-445. [2] Hoefler, B. C., et al,. Natl Acad Sci USA, 2012, 109, 13082-13087. [3] Moree, W. J., et al., Natl Acad Sci USA, 2012, 109, 13811-13816. [4] Debois, D., et al., J Am Soc Mass Spectrom. 2013, 24, 1202-1213 [less ▲]

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See detailAttribution of Cysteine Connectivities in small toxins - New Prospects Based on Partial Oxidation/Reduction Experiments and Ion-Mobility Mass Spectrometry
Quinton, Loïc ULg; Massonnet, Philippe ULg; Echterbille, Julien ULg et al

Conference (2013, December)

Disulfide bonds are post-translational modifications often found in biological compounds and especially in animal toxins. Disulfide bonds participate in the formation of specific folding of peptides and ... [more ▼]

Disulfide bonds are post-translational modifications often found in biological compounds and especially in animal toxins. Disulfide bonds participate in the formation of specific folding of peptides and proteins, directly related to their biological activity. Cystein pairing determinations are primordial for the synthesis of chemical homologous displaying the same bioactivity than the natural compound. This task appears already difficult when the cysteine pairings have to be determined from large proteins. The combination of physical and chemical techniques such as NMR, enzymatic proteolysis, liquid chromatography and mass spectrometry, is needed to circumvent this difficulty. However, when the work concerns small compounds such as conotoxins, the problem is much more complex due to the low amount of available compound and to the lack of enzymatic cleavage sites between cysteines. In this study, we investigate the case of small peptides that contain two disulphide bonds. The idea is to determine the cystein pairings in such compounds by a chemical partial reduction (or oxidation) of the peptides, followed by the separation of the generated species by ion-mobility mass spectrometry, and their characterisation by tandem mass spectrometry. Up to now, we have investigated the partial reduction not only in solution (with DTT and TCEP) but also in the gas-phase (Electron transfer dissociation), and partial oxidation in solution (with 3-CPBA). The results demonstrate an unexpected complexity of the data, including low fragmentation ratios of peptides and disulfide scramblings. [less ▲]

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See detailShort communication - Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere
Tarayre, Cédric ULg; Brognaux, Alison ULg; Bauwens, Julien ULg et al

in World Journal of Microbiology & Biotechnology (2013)

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2 ... [more ▼]

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacills subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, Bacillus subtilis strain ABGx produced xylanase and amylase. [less ▲]

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See detailPeptidomic comparison and characterization of the major components of the venom of the giant ant Dinoponera quadriceps collected in four different areas of Brazil.
Cologna, Camila Takeno; Cardoso, Jaqueline Dos Santos; Jourdan, Emmanuel et al

in Journal of Proteomics (2013), 94

Despite the noxious effects inflicted by Dinoponera ant's envenomation, the information about the biological properties and composition of their venom is still very limited. Ants from the genus Dinoponera ... [more ▼]

Despite the noxious effects inflicted by Dinoponera ant's envenomation, the information about the biological properties and composition of their venom is still very limited. Ants from the genus Dinoponera are believed to be the world's largest living ants with a body length of 3cm. Their occurrence is restricted to tropical areas of South America. In this work, we study the venom of the giant Dinoponera quadriceps ant collected in 4 different regions of Brazil. By using a combination of complementary mass spectrometric approaches, we aim at: (i) characterizing the venom composition of these ants; (ii) establishing a comparative analysis of the venom from four geographically different regions in Brazil. This approach demonstrates that ant venom is a copious source of new compounds. Several peptides were identified and selected for "de novo sequencing". Since most of the new peptides showed similarities with antimicrobial peptides (AMPs), antimicrobial assays were performed with the purpose of evaluating their activity. In regard to the comparative study of the four regions, we observed not only major differences in the venom compositions, but also that the venoms collected in closest areas are more similar than the ones collected in distant regions. These observations seem to highlight an adaption of the ant venoms to the local environment. Concerning the biological assays, the peptides called Dq-3162 and Da-3177 showed a wide-ranging antimicrobial activity. The characterization of new AMPs with a broad spectrum of activity and different scaffolds may aid scientists to design new therapeutic agents and understand the mechanisms of those peptides to interact with microbial membranes. The results obtained betoken the biotechnological potential of ant's venom. BIOLOGICAL SIGNIFICANCE: For the first time this manuscript describes an extensive proteomics characterization of the D. quadriceps venom. In addition this study reports the variation in venom composition of primitive ants from 4 geographically different areas of Brazil. The results reveal the presence of ~335 compounds for each venom/area and inter-colony variations were observed. 16 new peptides were characterized and 2 of them were synthesized and biologically assayed. These findings highlight the considerable and still unexplored diversity of ant's venom which could be used as valuable research tools in different areas of knowledge. [less ▲]

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See detailDer p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus
Herman, Julie ULg; Thelen, Nicolas ULg; Smargiasso, Nicolas ULg et al

in Biochimica et Biophysica Acta - General Subjects (2013), 1840

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation ... [more ▼]

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. Methods: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfermethod. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanismbymite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. Results: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. Conclusions: Der p 1 in either its recombinant formor in the natural context of house dustmite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. General significance: This finding suggests that Der p 1 may be valuable target against mites. [less ▲]

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See detailImaging mass spectrometry and proteomics of 3D cell cultures
Longuespée, Rémi ULg; Piron, Céline; Fléron, Maximilien et al

Poster (2013, October)

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See detailPolymer based intraocular lens adsorbome: a bottom up proteomics study
Bertrand, Virginie ULg; Huang, Yi-Shiang ULg; Mazzucchelli, Gabriel ULg et al

Conference (2013, September 08)

In the present work an optimized sample preparation protocol to identify and quantify the “adsorbomes” of hydrophilic and hydrophobic materials for IOLs known to have a higher or a lower incidence of PCO ... [more ▼]

In the present work an optimized sample preparation protocol to identify and quantify the “adsorbomes” of hydrophilic and hydrophobic materials for IOLs known to have a higher or a lower incidence of PCO, respectively was obtained. [less ▲]

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See detailStructural characterization of disulfide-bridged-peptides by a combined use of ETD, CID and Ion-Mobility mass spectrometry
Massonnet, Philippe ULg; Upert, Gregory; Pastor, Alexandra et al

Conference (2013, September 05)

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See detailProteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins
Sabri, Ahmed; Vandermoten, Sophie ULg; Leroy, Pascal et al

in PLoS ONE (2013), 8(9),

Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet ... [more ▼]

Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective. [less ▲]

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See detailUse of 1,5-diaminonaphthalene to combine matrix-assisted laser desorption/ionization in-source decay fragmentation with hydrogen/deuterium exchange
Lemaire, Pascale; Debois, Delphine ULg; Smargiasso, Nicolas ULg et al

in Rapid Communications in Mass Spectrometry [=RCM] (2013), 27(16), 1837-1846

In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the ... [more ▼]

In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the deuterons following in-solution hydrogen/deuterium exchange (HDX). This matrix must circumvent the commonly encountered undesired back-exchange reactions, in order to preserve the regioselective deuteration pattern. The 1,5-diaminonaphthalene (1,5-DAN) matrix is known to be suitable for MALDI-ISD fragmentation. MALDI Mass Spectrometry Imaging (MSI) was employed to compare 1,5-DAN and other commonly used MALDI matrices with respect to the extent of back-exchange and the uniformity of the H/D exchange profiles within the MALDI spots. We tested the back-exchange on the highly sensitive amyloid-beta peptide (1-40), and proved the regioselectivity on ubiquitin and b-endorphin. MALDI-MSI results show that 1,5-DAN leads to the least back-exchange over all the spot. MALDI-ISD fragmentation combined with H/D exchange using 1,5-DAN matrix was validated by localizing deuterons in native ubiquitin. Results agree with previous data obtained by Nuclear Magnetic Resonance (NMR) and Electron Transfer Dissociation (ETD). Moreover, 1,5-DAN matrix was used to study the H/D exchange profile of the methanol-induced helical structure of b-endorphin, and the relative protection can be explained by the polarity of residues involved in hydrogen bond formation. We found that controlling crystallization is the most important parameter when combining H/D exchange with MALDI. The 1,5-DAN matrix is characterized by a fast crystallization kinetics, and therefore gives robust and reliable H/D exchange profiles using MALDI-ISD. [less ▲]

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