Effet de la stabilité du courrant total d'ion en imagerie FT-ICR à harmonisation dynamique

Mass spectrometry imaging of complex biological samples often require high spectral resolution and mass accuracy to properly distinguish all isobaric compounds. To achieve such requirement dynamically harmonized FT-ICR analysers offer best results. However, mass accuracy below expectation due to masses shifting between pixels observed when performing image acquisitions. In this work we show a link between the mass shifting phenomenon andd the large variations observed in total ion current during image acquisitions. This work propose solve the mass shift by optimisation in sample preparation and acquisition parameters to stabilise the fluctuation of the total ion current in FT-ICR-MSI.EURLIPID

Peptide sequencing using gold nanoparticles in Surface-Assisted Laser Desorption/Ionization “in-source decay” mass spectrometry

The wide range of crucial functions fulfilled by biomolecules in biological systems can notably be attributed to the diversity in their structures. It is therefore essential to have effective methods for their structural characterization to support the understanding of their biological activities. In this context, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been extensively used for the analysis of the primary sequence of proteins and peptides. In particular, MALDI “in-source decay” (ISD) experiments can be used to fragment the molecular ions directly in the mass spectrometer ion source. Various matrices have already been studied in MALDI ISD MS experiments. However, the capabilities of surface-assisted laser desorption/ionization (SALDI) ISD MS, which employs nanostructured substrates (e.g. nanoparticles, solid nanosubstrates) instead of organic matrices, are still poorly documented. Here, we report SALDI ISD FT-ICR MS using citrate-coated gold nanoparticles (AuNPs). The “hardness” of the AuNPs was first evaluated based on the survival yield (SY) method with “thermometer ions”, and compared to commonly used MALDI matrices. The SY values acquired with the AuNPs and MALDI matrices were determined experimentally from the intensities of the parent and fragment benzylpyridinium thermometer ions, as follows : SY=I_Parent/(I_Parent+I_Fragment ) “Hard” assisting materials exhibit a low SY value (i.e. a high fragmentation yield). In this study, AuNPs turned out to be much harder than the studied MALDI matrices. The “hardness” ranking of the studied assisting materials was correlated with the initial ion velocity obtained by MALDI TOF MS and with previous “hardness” rankings. The SALDI mass spectra were characterized by a clean background in the low m/z range, facilitating the analysis of low molecular weight species and their fragments, due to the lack of interference caused by matrix ions and clusters. Then, the AuNPs were successfully tested in SALDI ISD MS for the sequencing of peptides, using an ESI/MALDI dual source 9.4T SolariX XR mass spectrometer, in the positive ionization mode. Several types of ISD fragment series (originating from sodium adducts of the molecular ion) were detected, offering a great sequencing coverage. As expected, c-ions produced via a radical-induced fragmentation pathway were detected. However, a- and y-ions were predominant in the mass spectra, suggesting that the fragmentation is mainly assisted by thermal ion activation. In conclusion, SALDI nanosubstrates offer interesting perspectives for the study of biopolymers, including peptides but also glycans and oligonucleotides, in SALDI ISD MS experiments

Label-free higher order structure and dynamic investigation method of proteins in solution using an enzymatic reactor coupled to electrospray high-resolution mass spectrometry

peer reviewe

Automatic metabolome profiling of bacterial colony heterogeneity by multimodal imaging with mass spectrometry and microscopy

peer reviewedIntroduction Mass Spectrometry Imaging (MSI) is a method of growing interest for the in-situ study of metabolites produced during bacterial colony interactions on hard surfaces. However, this type of analysis is often limited to one interaction per MS image or very few interactions. With this practice, the heterogeneity of different bacteria, i.e. the diversity in metabolites expression within the same bacterial strain, is not taken into account. Here, we propose a new informatic method that allows to study the molecular expression of multiple bacterial colonies from a single MS image by combining light microscopy and MALDI mass spectrometry imaging. The method automatizes the detection of different colonies and attributes signal from every detected metabolite to each colony. Methods Bacteria are grown on a thin layer of agar directly on an ITO (indium tin oxide) plate. A bright field microscopic image of the plate is taken before the MALDI preparation of the plate (pre-MALDI image). After MALDI MS acquisition of the plate a new bright field image of the plate is taken (post-MALDI image). A cross modality image registration is performed according to the MS, pre-MALDI and post-MALDI image. An image segmentation pipeline allows to determine the position of every detectable object on the plate. Then, the MS signal corresponding to each of those objects is estimated. This method generates a matrix of MS signal expression where rows correspond to the objects and columns to their metabolites. Preliminary Data We have applied our method on an agar coculture of Bacillus velezensis GA1 and Pseudomonas sp. CMR12a. Both strains are biocontrol agents that produce lipopeptides. The bacteria are inoculated on the opposite side of an ITO plate in such a way that the middle of the plate corresponds to the interaction between the 2 different strains. The bacteria are grown during 24 hours forming multiple micro-colonies of around 300 µm of diameter. Control plates are generated by inoculating each strain alone. The MSI of the plate is acquired on a MALDI FT-ICR-MS (SolariX XR 9.4T, Bruker) identifying different families of lipids such as phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and lipopeptides such as surfactins, orfamides and sessilins. The MS signal of each micro-colony is estimated according to our method and the corresponding image of each colony is extracted from the pre-MALDI image. The method detects multiple objects on the pre-MALDI image which is then filtered to isolate CMR12a colonies. The removed objects correspond to GA1 colonies and artefacts with not enough signal to be analyzed. It is then possible to investigate the heterogeneity of the colonies by applying unsupervised clustering algorithms (hierarchical clustering). Statistical analysis is used to detect specific signals of those clusters. In this experiment, our method highlights a sub-cluster of CMR12a which is characterized by an over expression of particular PG lipids and an under expression of sessilins and orfamides. The colonies corresponding to this sub-cluster seems to be located closer to the interaction region with GA1 compared to the other CMR12a colonies. Moreover, the method highlights multiple CMR12a mutants previously identified as mutants that have lost a genomic island which over expressed PE lipids and do not express sessilins. Further work will focus on in vivo analysis of bacteria colonizing plant’s roots. Novel Aspect Informatic method allowing the automatic detection and the study of multiple bacterial micro-colonies by their MS and microscope image

Investigation of structure-stabilizing elements in proteins by ion mobility mass spectrometry and collision-induced unfolding

peer reviewe

Lipopeptides as rhizosphere public goods for microbial cooperation.

peer reviewedHere, we provide new insights into the possible fate of cyclic lipopeptides as prominent specialized metabolites from beneficial bacilli and pseudomonads once released in the soil. Our data illustrate how the B. velezensis lipopeptidome may be enzymatically remodeled by Streptomyces as important members of the soil bacterial community. The enzymatic arsenal of S. venezuelae enables an unsuspected extensive degradation of these compounds, allowing the bacterium to feed on these exogenous products via a mechanism going beyond linearization, which was previously reported as a detoxification strategy. As soils are carbon-rich and nitrogen-poor environments, we propose a new role for cyclic lipopeptides in interspecies interactions, which is to fuel the nitrogen metabolism of a part of the rhizosphere microbial community. Streptomyces and other actinomycetes, producing numerous peptidases and displaying several traits of beneficial bacteria, should be at the front line to directly benefit from these metabolites as "public goods" for microbial cooperation

Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition

From FMTV to WATERS: Lessons Learned from the First Verification Challenge at ECRTS

We present here the main features and lessons learned from the first edition of what has now become the ECRTS industrial challenge, together with the final description of the challenge and a comparative overview of the proposed solutions. This verification challenge, proposed by Thales, was first discussed in 2014 as part of a dedicated workshop (FMTV, a satellite event of the FM 2014 conference), and solutions were discussed for the first time at the WATERS 2015 workshop. The use case for the verification challenge is an aerial video tracking system. A specificity of this system lies in the fact that periods are constant but known with a limited precision only. The first part of the challenge focuses on the video frame processing system. It consists in computing maximum values of the end-to-end latency of the frames sent by the camera to the display, for two different buffer sizes, and then the minimum duration between two consecutive frame losses. The second challenge is about computing end-to-end latencies on the tracking and camera control for two different values of jitter. Solutions based on five different tools - Fiacre/Tina, CPAL (simulation and analysis), IMITATOR, UPPAAL and MAST - were submitted for discussion at WATERS 2015. While none of these solutions provided a full answer to the challenge, a combination of several of them did allow to draw some conclusions

Exploring the Phytobeneficial and Biocontrol Capacities of Endophytic Bacteria Isolated from Hybrid Vanilla Pods.

peer reviewedIn this study, 58 endophytic bacterial strains were isolated from pods of two hybrid vanilla plants from Madagascar, Manitra ampotony and Tsy taitra. They were genetically characterized and divided into four distinct phylotypes. Three were associated to genus Bacillus species, and the fourth to the genus Curtobacterium. A selection of twelve strains corresponding to the identified genetic diversity were tested in vitro for four phytobeneficial capacities: phosphate solubilisation, free nitrogen fixation, and phytohormone and siderophore production. They were also evaluated in vitro for their ability to biocontrol the growth of the vanilla pathogenic fungi, Fusarium oxysporum f. sp. radicis vanillae and Cholletotrichum orchidophilum. Three bacteria of phylotype 4, m62a, m64 and m65, showed a high nitrogen fixation capacity in vitro, similar to the Pseudomonas florescens F113 bacterium used as a control (phospate solubilizing efficiency respectively 0.50 ± 0.07, 0.43 ± 0.07 and 0.40 ± 0.06 against 0.48 ± 0.03). Strain t2 related to B. subtilis showed a higher siderophore production than F113 (respectively 1.40 ± 0.1 AU and 1.2 ± 0.1 AU). The strain m72, associated with phylotype 2, showed the highest rate of production of Indole-3-acetic acid (IAA) in vitro. Bacteria belonging to the pylotype 4 showed the best capacity to inhibit fungal growth, especially the strains m62b m64 and t24, which also induced a significant zone of inhibition, suggesting that they may be good candidates for controlling fungal diseases of vanilla. This competence was highlighted with spectral imaging showing the production of lipopeptides (Iturin A2 and A3, C16 and C15-Fengycin A and C14 and C15-Surfactin) by the bacterial strains m65 confronted with the pathogenic fungi of vanilla.FEDER BIOMED HUB Technology Suppor