MALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens

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

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 ▲]

Discrimination of Isobaric Leu/Ile Residues by MALDI In-source Decay Mass Spectrometry

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

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 ▲]

Structural characterization of disulfide-bridged-peptides by a combined use of ETD and Ion-Mobility mass spectrometry

Poster (2013, May 03)

Development of an analytical method to detect metabolites of nitrofurans. Application to the study of furazolidone elimination in Vietnamese black tiger shrimp (Penaeus monodon)

in Aquaculture (2013), 376-379

Nitrofurans, banned antibiotics in European Union (EU), have often been found in imported aquaculture products in the past and are still found nowadays according to the Rapid Alert System for Feed and ... [more ▼]

Nitrofurans, banned antibiotics in European Union (EU), have often been found in imported aquaculture products in the past and are still found nowadays according to the Rapid Alert System for Feed and Food (RASFF) of the European Commission. A quantitative method based on liquid chromatography coupled to isotopic dilution tandem mass spectrometry (LC–IDMS/MS) was developed for the determination of the residues of four nitrofuran antibiotic residues in shrimps. The experimental protocol consisted of an acid-catalysed release of protein-bound metabolites, followed by derivatisation with 2-nitrobenzaldehyde (NBA). Then, a double liquid–liquid extraction with ethyl acetate was performed before LC–IDMS/MS analysis by positive electrospray ionisation (ES+) with multiple reaction monitoring (MRM) of two transitions per compound. An “in-house” validation of the method for shrimp analysis was conducted according to the EU criteria for the analysis of veterinary drug residues in foods. The decision limits (CCalpha) were 0.08–0.36 µg kg -1 and the detection capabilities (CCbeta) were 0.12–0.61 µg kg-1, which are both below the minimum required performance limit (MRPL) set at 1 µg kg -1 by the EU. The developed method was applied to evaluate the elimination of furazolidone residues in shrimp muscles after a contamination experiment. After 28 days of decontamination, a concentration of 115 µg kg-1 of furazolidone metabolite 3-amino-2-oxazolidinone (AOZ) was still measured in shrimp muscle. [less ▲]

Influence of lignin in Reticulitermes santonensis: symbiotic interations investigated through proteomics

in Symbiosis (2013)

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting ... [more ▼]

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting together and with the insect host. Their likely ability to degrade ligno-cellulosic compounds could lead to improvements in second generation biofuels production. Lignin elimination represents a critical point as this polymer significantly interferes with industrial process of cellulose. Although host produces its own lignin-degrading enzymes, some symbionts may participate in digestion of lignin and its degradation products in termite gut. Here, we compared gut proteomes from R. santonensis after rearing on artificial diets composed of cellulose with and without lignin. The effect of lignin in artificial diets on different parts of the digestive tract was compared through liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) experiments. Enzymatic assays were performed to characterize activities present in R. santonensis digestive tract after feeding on artificial diets. Microscopic observations of microbial communities provided some information on population balances after feeding experiment. [less ▲]

Effects of a sublethal pesticide exposure on locomotor behavior: A video-tracking analysis in larval amphibians

in Chemosphere (2013), 90(3), 945-951

Organochlorine pesticides such as endosulfan have been shown to have both lethal and sublethal effects on amphibians. In this context, behavioral endpoints have proved their usefulness in evidencing ... [more ▼]

Organochlorine pesticides such as endosulfan have been shown to have both lethal and sublethal effects on amphibians. In this context, behavioral endpoints have proved their usefulness in evidencing impacts of such chemicals at environmental concentrations that do not necessarily cause mortality. The recent development of video-tracking technologies now offers the possibility of accurately quantifying locomotor behaviors. However, these techniques have not yet been applied to evaluating the toxicity of pesticides in amphibians. We therefore aimed at determining the potential toxicity of endosulfan on endpoints associated with locomotion after short-term environmental endosulfan exposure in Rana temporaria tadpoles and at using these data as warning systems for survival alterations after a longer exposure. To this end, we analyzed video-tracks of 64 tadpoles (two pesticide treatments: 5 and 50 μg L−1, one control and one solvent-control) with Ethovision XT 7 software. The highest endosulfan concentration had a significant effect on all four behavioral endpoints. Contaminated tadpoles traveled shorter distances, swam less often, at a lower mean speed, and occupied a less peripherical position than control tadpoles. The lowest endosulfan concentration had similar but lower effects, and did not affect mean speed during swimming. Survival was reduced only after a long-term exposure to endosulfan and was associated with short-term behavioral dysfunctions. These results show that endosulfan strongly affects the behavioral repertory of amphibian tadpoles, but in different ways depending on concentration, thus suggesting that the pesticide has complex modes of action. Given the importance of locomotion and space use in tadpole success in their aquatic environment, these results confirm the toxic action of endosulfan. By highlighting effects before mortality markers, video-tracking systems also show their potential as sentinels of sublethal effects of pesticides. [less ▲]

Proteomic investigation of aphid honeydew reveals an unexpected diversity of proteins

in PLoS ONE (2013), Accepted pending revision

Preliminary assessment of the risk linked to furan ingestion by babies consuming only ready-to-eat food

in Food Additives & Contaminants. Part A. Chemistry, Analysis, Control, Exposure & Risk Assessment (2013)

The risk linked to furan ingestion has been assessed in previous papers for Belgian adults and children (Scholl et al., 2012b; Scholl et al., 2012c). The present paper focuses on infants consuming only ... [more ▼]

The risk linked to furan ingestion has been assessed in previous papers for Belgian adults and children (Scholl et al., 2012b; Scholl et al., 2012c). The present paper focuses on infants consuming only ready-to-eat baby food. As there is no Belgian baby dietary database, the furan exposure assessment was carried out by using Italian infant consumption database and Belgian contamination data. The estimated daily intake (EDI) was calculated according to a deterministic methodology. It involved 42 commercially available ready-to-eat baby food and 36 baby consumption records. The mean EDI was 1,460 ng * (kgb.w.*day)-1 which is 3.8 times higher than the 381 ng * (kgb.w.*day)-1 reported for Belgian adults, and 3.5 times higher than the 419 ng * (kgb.w. * day)-1 measured for Belgian children. To assess and characterize the risk for babies exposure the Margin of Exposure (MoE) was calculated. It highlighted that 74% of infants have a MoE below 1,000, with a minimum of 140. However, these are only preliminary results as they were calculated from a very small dataset and the infant cytochrome P450 activity is significantly different compared to the adult. Therefore, the risk linked to furan ingestion by babies should be assessed in a different manner. To this end, additional data regarding a baby diet as well as a better understanding of furan toxicity for babies are needed to characterize more accurately the risk for infants. [less ▲]

Ion-Mobility mass spectrometry as a potential tool to assign disulfide bonds arrangements in peptides with multiple disulfide bridges.

in Analytical Chemistry (2013)

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually ... [more ▼]

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using MS/MS spectra of the totally reduced unfolded species but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of non-classical ions. MS/MS alone does not allow the cysteine pairing nor the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining Ion Mobility Spectrometry (IMS)and Collision Induced Dissociation(CID). It is assumed that the opening of one S-S bridges in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence, but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteines connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference. [less ▲]

Secretion and maturation of conotoxins in the venom ducts of Conus textile

in Toxicon (2012), 60(8), 1370-1379