Analysis of Sugar Component of a Hot Water Extract from Arabidopsis thaliana Pollen Tubes Using GC-EI-MS

International audienceExtraction with hot water is the oldest and simplest method used to recover pectin from an alcohol insoluble residue extract, although this method has not been widely used for the cell wall analysis of pollen tube, a model used to study cell wall. This protocol described this method applied for pectin extraction from 6 h-old Arabidopsis pollen tubes followed by a sugar composition analysis by gas chromatography mass spectrometry

User-friendly extraction and multistage tandem mass spectrometry based analysis of lipid-linked oligosaccharides in microalgae

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Cell wall biochemical alterations during Agrobacterium -mediated expression of haemagglutinin-based influenza virus-like vaccine particles in tobacco

International audienceInfluenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing

From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in Insects

One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects

Use of procaine and procainamide as derivatizing co-matrices for the analysis of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

International audienceRATIONALE: Analysis of oligosaccharides by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry often yields only alkali metal cation adducts, which results in lower fragmentation yields and difficulty to retrieve sequence information. Derivatization by reductive amination may be used to promote Y-type glycosidic cleavages. However, this involves time-consuming preparations and purifications with sample loss. Here, procaine and procainamide were used directly as co-matrices with 2,5-dihydroxybenzoic acid (DHB). METHODS: Acidified 10 g/L procaine hydrochloride or procainamide hydrochloride solutions in water/acetonitrile were added to the oligosaccharide solution one minute before preparing our MALDI targets using DHB with the dried-droplet method. This simple protocol resulted in deposits of very fine homogeneous crystals. RESULTS: Positive ion mass spectra, easily acquired in an automated mode, presented a high percentage of oligosaccharides derivatized as Schiff base or glycosylamine notably detected as protonated molecules [M + H](+). The high abundance of procaine or procainamide on the target did not impede the ionization process, improved the signal-to-noise ratio and eliminated the need to search for 'sweet spots'. Fragmentation of the protonated precursor ions of the derivatives largely favored Y-type glycosidic cleavages. CONCLUSIONS: This easy and fast sample preparation, involving low toxicity and easily accessible chemicals, allowed the selection of protonated molecules as precursor ions for post-source decay analyses. This opened the possibility of simplifying sequence retrieval in routine oligosaccharide analyses

Dendrimères polyamidoamines : synthèse, caractérisation structurale et étude de complexes non covalents polyamidoamines / ADN

Ces travaux de thèse s articulent autour de trois grands axes de recherche : snthèse et modification chimique des deux classes de PAMAMs : la synthèse des PAMAMs a été réalisée selon l approche divergente de Tomalia. Les modifications chimiques des fonctions de surface ont impliqué le greffage d acides aminés - phénylalanine, histidine et glycine- ou de groupements phényle, de telles modifications pouvant moduler leur bioactivité et biocompatibilité. Les PAMAMs synthétisés et modifiés ont été caractérisés par spectrométrie de masse (MS), spectrométrie de mobilité ionique couplée à la MS (IM-MS) ainsi que par résonance magnétique nucléaire. Etude des structures défectueuses des PAMAMs : la voie de synthèse choisie génère, outre les produits attendus, divers produits secondaires. La séparation, l identification et la quantification de ces structures défectueuses ont été appréhendées par couplage de la MS avec diverses techniques séparatives dont l électrophorèse capillaire, la chromatographie liquide à interaction hydrophile, la chromatographie sur couche mince et la spectrométrie de mobilité ionique. L apport, la complémentarité et les limites de chaque approche sont discutés. Etude des complexes non-covalents PAMAM/ADN : les stœchiométries, stabilités, constantes de dissociation des complexes ainsi que l affinité des ligands pour l ADN ont été déterminées pour divers dendriplexes par ESI-MS et IM-MS. L intérêt de l IM-MS est ici démontré pour séparer, mettre en évidence les différentes espèces présentes dans les échantillons (dendriplexes, simple-brin, duplexe et quadruplexe d ADN, etc ), diminuer la complexité des spectres et améliorer la qualité spectrale.This thesis is composed of three main research areas: Synthesis and chemical modification of PAMAMs: PAMAMs were synthetized according to the divergent approach of Tomalia. In order to modulate their bioactivity and biocompatibility, chemical modifications of the surface end-groups were carried out by grafting amino acids - phenylalanine, histidine and glycine- or phenyl groups. Native and modified-PAMAMs were characterized by mass spectrometry (MS), ion mobility spectrometry coupled to MS (IM-MS) and nuclear magnetic resonance (NMR). Study of defective structures of PAMAMs: the divergent synthesis generated, in addition to the expected products, various byproducts that needed to be identified and characterized. The separation, identification and quantification of these defective structures were achieved by coupling MS with various separation techniques: capillary electrophoresis, hydrophilic interaction liquid chromatography, thin layer chromatography or the ion mobility spectrometry. The contribution, complementarity and limits of each approach are discussed. Study of non-covalent complexes PAMAM/DNA: the stoichiometries, stabilities, dissociation constants of complexes were determined by ESI-MS and IM-MS for various dendriplexes. IM-MS was found particularly useful to separate and highlight the different species present in the samples (dendriplexes, DNA duplex and quadruplex, single-stranded DNA, etc ...), reduce the complexity of the spectra and improve spectral quality.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Mass Spectrometry and Ion mobility as a Tool to Investigate Reaction Pathways in Organic Chemistry and more

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Stand reaction of linseed oil

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A solventless synthesis process of new UV-curable materials based on linseed oil

International audienceA new synthesis process of polymer materials based on vegetable oil has been developed. First, linseed oil was thermally polymerized in bulk at 300 °C under an inert atmosphere. Next, the obtained stand oil was functionalized in a two-step one-shot process without solvent in order to graft on some photopolymerizable groups. The first step consisted of a bulk reaction with maleic anhydride at 220 °C under an inert atmosphere. The grafted anhydride groups were then used in the second step as an anchor for grafting 2-hydroxyethylmethacrylate at room temperature. Finally, the materials were prepared by UV-curing of the modified linseed oil and then characterized. The obtained materials are flexible, hydrophobic and not biodegradable, and may have potential applications in flexible coatings