References of "Smargiasso, Nicolas"
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See detailSpatiotemporal monitoring of the antibiome secreted by Bacillus biofilms on plant roots using MALDI mass spectrometry imaging
Debois, Delphine ULg; Jourdan, Emmanuel; Smargiasso, Nicolas ULg et al

in Analytical Chemistry (2014), 86(9), 4431-4438

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The ... [more ▼]

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The plant protective activity of these bacteria has been correlated with the potential to secrete a wide array of antibiotic compounds upon growth as planktonic cells in isolated cultures under laboratory conditions. However, in situ expression of these antibiotics in the rhizosphere where bacterial cells naturally colonize root tissues is still poorly understood. In this work, we used Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) to examine spatio-temporal changes in the secreted antibiome of B. amyloliquefaciens developing as biofilms on roots. Non-ribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time-frames and quantities in the surrounding medium. Interestingly, MS/MS experiments performed directly from the gelified culture medium, also allowed to identify a new variant of surfactins released at later time points. However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. This first dynamic study reveals the power of MALDI MSI as tool to identify and map antibiotics synthesized by root-associated bacteria and more generally, to investigate plant-microbe interactions at the molecular level. [less ▲]

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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 detailInfluences of proline and cysteine residues on fragment yield in matrix-assisted laser desorption/ionization in-source decay mass spectrometry
Asakawa, Daiki; Smargiasso, Nicolas ULg; Quinton, Loïc ULg et al

in Journal of the American Society for Mass Spectrometry (2014)

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See detailComparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients.
Quesada-Calvo, Florence; Bertrand, Virginie ULg; Longuespée, Rémi ULg et al

in Journal of proteomics (2014), 112C

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic ... [more ▼]

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work. [less ▲]

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See detailTissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology
Longuespée, Rémi ULg; Fléron, Maximilien; Pottier, Charles et al

in OMICS : A Journal of Integrative Biology (2014)

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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 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 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 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 detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel; Smargiasso, Nicolas ULg et al

Poster (2013, June 12)

Some non-pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non-pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment.Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were incubated at 28°C with a 16h photoperiod. Different growth / incubation durations were studied: 10/3; 13/7; 21/14 and 39/32. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. (HCCA, 5mg/mL in ACN/0.2% TFA 70:30) was used as matrix. UltraFlex II TOF/TOF and Solarix FT-ICR mass spectrometers were used to record molecular cartographies and perform MS/MS experiments for structural analysis purposes. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 10/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Tandem mass spectrometry experiments, performed on the dried culture medium, allowed to partially sequence these new lipopeptides. MS/MS results allied to exact mass measurements and isotopic pattern simulation give good confidence in the chemical structure we suggest. Nevertheless, to fully identify these new variants of surfactin, micro-extractions followed by (LC)-nano-ESI-MS/MS using a LESA module are in progress. MALDI Mass Spectrometry Imaging becomes a tool to decipher inter-species molecular communication. [less ▲]

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See detailDiscrimination of Isobaric Leu/Ile Residues by MALDI In-source Decay Mass Spectrometry
Asakawa, Daiki; Smargiasso, Nicolas ULg; De Pauw, Edwin ULg

in Journal of the American Society for Mass Spectrometry (2013), 24(2), 297-300

MALDI in-source decay (ISD) has been used for the top-down sequencing of proteins. The use of 1,5-diaminonapthalene (1,5-DAN) gave strong intensity of w ions, which are informative fragments and can be ... [more ▼]

MALDI in-source decay (ISD) has been used for the top-down sequencing of proteins. The use of 1,5-diaminonapthalene (1,5-DAN) gave strong intensity of w ions, which are informative fragments and can be helpful for the distinction of the isobaric amino acids, Leu and Ile. Our data suggests that the w fragments are formed from z* radical fragment by unimolecular dissociation and high abundance of w ions in MALDI-ISD with 1,5-DAN can be understood as resulting from the low collision rate in the MALDI plume. The MALDI-ISD with 1,5-DAN could be a useful method for the top-down sequencing of proteins including discrimination of Leu and Ile near the C-terminal end. [less ▲]

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