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<p>Lipids are important cellular biomolecules that perform essential functional and biological roles. For instance, lipids in the cell are the compartmentalizer for the cytoplasm and an energy storage unit. The knowledge surrounding lipids is abundant, yet there is still so much to uncover. There are many categories of lipids and within each category the structural composition is extremely diverse. In turn, the dramatic structural complexity of lipids demands analytical methods capable of providing in-depth structural characterization of individual molecular structures. However, lipid structural elucidation has remained challenging, namely due to the presence of isomeric and isobaric species with a complex mixture. In particular, isomeric/isobaric lipid structures arise from variations in class, headgroup, fatty acyl chain, <i>sn</i>-position, and/or carbon-carbon double bond (C=C) position(s). Recently, recent research suggests C=C composition impacts lipid physical properties, metabolic fate, and intermolecular interactions. Thus, analytical strategies capable of localizing sites of unsaturation are of interest in the lipidomics community.</p> <p>Mass spectrometry (MS) is a leading tool for lipid analysis. Electrospray ionization (ESI), a soft ionization method, is the most commonly used method for lipid ionization as a means of taking the ions from liquid-phase to gas-phase without extensive decomposition of the species. Utilizing ESI-MS, lipids can be identified at a sum compositional level via accurate mass measurements. . With tandem mass spectrometers, lipid ions can be further probed, utilizing tandem-MS (MS/MS) to generate structurally informative product ion spectra that facilitate the assignment of lipid molecular structure. More so, gas-phase ion/ion reactions represent a unique MS-based technique that has improved the analysis of lipids structures. Gas-phase ion/ion reactions allow for lipid species to be charge inverted from one polarity to the opposite polarity. This reaction enables lipids to be ionized in a polarity that is optimal for class identification and further investigated in the opposite polarity where more structural information is obtained. All the information provided is captured without the requirement of multiple solution conditions which is necessary when analyzing in both polarities. In the case of charge inverted lipids from positive ion mode to negative ion mode, fatty acyl composition can be obtained; however, C=C information is lacking.</p> <p>MS can also be paired with other analytically technologies to assist with lipid analysis. One of those technologies is liquid chromatography (LC), which allows for the separation of lipids based on different characteristic depending on the column type being used. Reverse-phase LC (RPLC) allows for the separation of lipid molecular species based on structural composition. RPLC-MS/MS benefits from the ability to separate lipids and determine their fatty acyl chain composition but it is difficult to specify C=C location with the use of a synthetic standard that is identical to each molecular species being analyzed.</p> <p>Commonality between the gas-phase ion/ion reactions for charge inversion of lipids and RPLC-MS/MS approaches is the inability to provide C=C coverage. In-solution and unique ion activation techniques have been developed for seeking such information. The Paternò–Büchi reaction is a UV-initiated [2 + 2]-cycloaddition of an excited carbonyl containing compound onto an olefin group. This reaction can be initiated onto the alkene group within an unsaturated lipid aliphatic chain to form an oxetane ring modification. There are two product ions that can be formed upon each unsaturation site due to a lack of regioselectivity the reagent can attach at either side of the C=C. The modified lipids can be taken into gas-phase and collisionally activated via low-energy collision induced dissociation, generating product ions indictive of C=C position(s). The work herein shows the incorporation of the PB reaction into the gas-phases ion/ion reaction and RPLC-MS/MS apparatuses for C=C localization. The methods have been applied to the lipid extracts of bovine liver and human plasma for confident molecule species determination.</p>

Analyse fonctionnelle de la protéine Enhancer of Zeste, SlEZ2, chez la tomate, Solanum lycopersicumLes protéines Polycomb, initialement découvertes chez la drosophile, ont récemment caractérisées chez les plantes où elles remplissent des fonctions essentielles au cours du développement de la plante. Chez la drosophile, les protéines polycomb (PcG) agissent sous forme de trois complexes multi-protéiques : PRC1, PRC2 et PhoRC. Seulement, deux de ces complexes ont été identifiés chez les plantes : un orthologue fonctionnel du complexe PRC1 (PRC1-like) et PRC2. Le complexe PRC2 maintien la chromatine dans un état condensé et intervient dans le contrôle du développement des fleurs, des graines, des fruits et des feuilles. Chez la tomate Solanum lycopersicum, le complexe PRC2 est composé de trois protéines polycomb : SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste). Les protéines SlE(Z) portent l’activité histone méthyl transférase qui permet la mise en place de la marque répressive H3K27me3. Chez la plante modèle, Arabidopsis thaliana, cette marque joue un rôle essentiel au cours du développement de la plante Afin d’étudier le rôle du complexe PRC2 dans le développement du fruit et de la plante de tomate, et plus particulièrement de la protéine SlE(Z), nous avons identifié trois gènes codant les protéines SlE(Z) : SlEZ1, SlEZ2 et SlEZ3. Au laboratoire, il a récemment été montré que la protéine SlEZ1 intervient au cours du développement floral (How Kit et al., 2010). L’objectif de ce travail est de déterminer la fonction de la protéine SlEZ2 au cours du développement du fruit et de la plante de tomate. Pour cela, nous avons analysé des plantes transgéniques sous exprimant le gène SlEZ2, orthologue au gène CURLY LEAF d’A. thaliana, par stratégie RNAi. Ce travail indique que la protéine SlEZ2 est impliquée dans la croissance de la plante de tomate, ainsi que dans le développement des feuilles, des fleurs et des fruits. Les plantes transgéniques présentent des phénotypes pléiotropes tels que des fleurs et des feuilles modifiées, un fort taux d’avortement des fruits, des fruits de texture et de couleur altérées ainsi qu’une réduction de la taille des plantes. De plus, nous avons identifiés quatre gènes ciblés par la protéine SlEZ2 dont l’expression est dérégulée dans les feuilles. Il s’agit de deux gènes à MADS box, TAG1 et TAGL1, ainsi que de deux gènes KNOX, LeT6 et TKN4. / Functional analysis SlEZ2, a tomato Enhancer of zeste proteinPolycomb proteins, first discovered in Drosophila, have been identified in plants and play essential functions in plant development. In Drosophila, polycomb proteins (PcG) acts as a complex and three have been identified: PRC1, PRC2 and PhoRC. However, only two polycomb complexes have been identified in plants: like-PCR1 and PRC2. The PCR2 complex maintain chromatin in a closed state and control flower, seed, fruit and leaf development.In tomato Solanum lycopersicum, PRC2 is composed by three polycomb proteins SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste)(Enhancer of Zeste). SlE(Z) proteins have a methyltransferase activity that puts in place an repressive epigenetic mark a trimethylation of lysine 27 histone 3. In plant model, Arabidopsis thaliana, this mark plays an essential role in plant development but little is known about PRC2 role in plant and fruit development of tomato. In order to unravel the function of the E(z) protein in the control of tomato fruit and plant development, we have characterized three E(z) encoding genes, namely SlEz1, SlEz2 and SlEZ3. In a recent work, we reported that SlEZ1 protein plays a role in flower development (How Kit at al., 2010). The aim of this present study was to determine the function of the SlEZ2 protein in plant and fruit development. We present our results focusing on RNAi transgenic plants which underexpressed SlEZ2 gene, homologue of Curly Leaf Arabidopsis gene. This analysis indicates that SlEZ2 protein is implicated in tomato plant growth and affects also leaf, flower and fruit development. Phenotypes include abnormal flowers and leafs, fruit development abortion, altered fruit colour and texture and plant of reduced size. Moreover, we characterize four target genes of SlEZ2 genes in leaves which present a deregulated expression : TAG1, TAGL1, LeT6 and TKN4.

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