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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 detailProjet Termitofuel: les termites et leurs symbiontes pour mieux valoriser la biomasse ligno-cellulosique
Bauwens, Julien ULg; Tarayre, Cédric ULg; Brasseur, Catherine ULg et al

Poster (2013, October 19)

L’exploitation actuelle de la biomasse ligno-cellulosique, extrêmement abondante sur Terre, est liée à la production de déchets relativement peu valorisables. C’est pourquoi le projet TERMITOFUEL étudie ... [more ▼]

L’exploitation actuelle de la biomasse ligno-cellulosique, extrêmement abondante sur Terre, est liée à la production de déchets relativement peu valorisables. C’est pourquoi le projet TERMITOFUEL étudie la digestion du bois chez les termites, grâce à une approche pluridisciplinaire, en vue d’améliorer la production de bioéthanol de seconde génération. Au sein de l’Unité d’Entomologie Fonctionnelle et Evolutive, l’approche protéomique est employée afin d’identifier les micro-organismes présents dans le tube digestif des termites et caractériser les activités enzymatiques. [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 <br />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 <br />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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See detailMALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens
Debois, Delphine ULg; Ongena, Marc ULg; Cawoy, Hélène ULg et al

in Journal of the American Society for Mass Spectrometry (2013), 24(8), 1202-1213

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial ... [more ▼]

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDIFTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology. [less ▲]

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See detailIn-Source Decay during Matrix-Assisted Laser Desorption/Ionization Combined with the Collisional Process in an FTICR Mass Spectrometer
Asakawa, Daiki; Calligaris, David; Zimmerman, Tyler et al

in Analytical Chemistry (2013), 85

The type of ions detected after in-source decay (ISD) in a MALDI source differs according to the ion source pressure and on the mass analyzer used. We present the mechanism leading to the final ISD ions ... [more ▼]

The type of ions detected after in-source decay (ISD) in a MALDI source differs according to the ion source pressure and on the mass analyzer used. We present the mechanism leading to the final ISD ions for a Fourier transform-ion cyclotron resonance mass spectrometer (FTICR MS). The MALDI ion source was operated at intermediate pressure to cool the resulting ions and increase their lifetime during the long residence times in the FTICR ion optics. This condition produces not only c′, z′, and w fragments, but also a, y′, and d fragments. In particular, d ions help to identify isobaric amino acid residues present near the Nterminal amino acid. Desorbed ions collide with background gas during desorption, leading to proton mobilization from Arg residues to a less favored protonation site. As a result, in the case of ISD with MALDI FTICR, the influence of the Arg residue in ISD fragmentation is less straightforward than for TOF MS and the sequence coverage is thus improved. MALDI-ISD combined with FTICR MS appears to be a useful method for sequencing of peptides and proteins including discrimination of isobaric amino acid residues and site determination of phosphorylation. Additionally we also used new software for in silico elimination of MALDI matrix peaks from MALDI-ISD FTICR mass spectra. The combination of high resolving power of an FTICR analyzer and matrix subtraction software helps to interpret the low m/z region of MALDI-ISD spectra. Finally, several of these developed methods are applied in unison toward a MALDI ISD FTICR imaging experiment on mouse brain to achieve better results. [less ▲]

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See detailInnovative analytical strategy using ion-mobility shifting additive for isobaric selenium compound identification in selenomethionine standards by IMS
Kune, Christopher ULg; Far, Johann ULg; Eppe, Gauthier ULg et al

Conference (2013, July 09)

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Selenomethionine (SeMet) is one of the widely used selenium standard during ... [more ▼]

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Selenomethionine (SeMet) is one of the widely used selenium standard during Selenium speciation studies. This work was focused on the elaboration of an analytical strategy for the detection and the structural elucidation of an isobaric Se interference, which is found in standard solutions of SeMet by high resolution mass spectrometry (Rm/Δm > 20.000). The structural elucidation of these compounds requires the isolation of the respective parent ion. Nevertheless, the mass difference between SeMet and its interference is less than 0.02Da which is well below the window selection of conventional techniques in mass spectrometry (Quadripole, ion trap). The empirical formula and double bound equivalent (DBE) of these ions suggest different tridimensional structures which lead to a discrimination depending on the ion mobility. This separation is observed, both in gaseous and liquid phase, by Ion Mobility Spectrometry (IMS), Capillary Electrophoresis (CE) and Liquid Chromatography (LC) which are hyphenated to mass spectrometry as detector. The separation efficiency of these ions by IMS and CE is improved by using specific shifting agents (18-Crown-6 Ether) selective to only one of these ions. This strategy has successfully separated the two isobaric ions present and leads to the structural elucidation of the isobar contaminant of SeMet. [less ▲]

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See detailInnovative analytical strategy using ion-mobility for structural or functional selenium isomers identification by ion mobility spectrometry
Far, Johann ULg; Kune, Christopher ULg; Lobinski, Ryszard et al

Poster (2013, July)

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Se-rich yeast is one of the most popular Se source for supplementation. The ... [more ▼]

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Se-rich yeast is one of the most popular Se source for supplementation. The classical method of speciation is related to multidimensional liquid chromatography (LC) hyphenated to mass spectrometry (MS) Recent advances in Se speciation led to greatly improve the Se speciation in these samples but isomers identification and quantification remain challenging. This work focuses on the elaboration of an innovative analytical strategy for the detection and the structural elucidation of isobaric selenium compounds present in Se-rich yeast. A specific complex formation agent acts as a chemical probe for the detection of chemical function. The addition of a complexing agent can improve the discrimination between structural or functional Se isomers using ion mobility techniques as Ion Mobility Spectrometry (IMS) by increasing the molecular weight (i.e. the m/z ratio in MS) and the collision cross section of a target ion after selective complexation. This Ion Mobility orthogonal separation improves the structural elucidation. Crown ethers used as shifting agents can specifically form complexes with primary amines. The addition of crown ether to different low molecular weight fractions obtained by multidimensional LC of a water extract from Se-rich yeast permitted to detect Se isomers and confirmed their structure using IMS. [less ▲]

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