FRAGMENTATION DANS UNE SOURCE A ÉLECTRONÉBULISATION

VIGNY P., Professeur des Universités détaché au CNRS : Président LANGE C., Professeur, Université de Rouen : Co-Directeur DELMAS A., Directeur de recherche CNRS Orléans :Co-Directeur TORTAJADA J., Directeur de recherche CNRS Université d'Evry Val d'Essonne : Rapporteur LAPREVOTE O., Directeur de recherche CNRS Gif sur Yvette : Rapporteur LAFOSSE M., Professeur, Universitéd'Orléans : ExaminateurThis work dealt with the study of peptides and oligonucleotides by in-source fragmentation with a Quattro II (Micromass). This instrument was first evaluated in the positive mode with peptide thioesters and then in the negative mode with brominated oligodeoxynucleotides whose fragmentation of the modified base allows its location in the sequence. The fragmentation of peptide acetals and diols (hydrated aldehydes) leads to a same cyclic final ion. The hydration of peptide aldehydes was studied in solution by NMR and in the gas phase by in-source fragmentation. The information coming from this work allowed a better understanding of the mechanism of the ligation chemistry by oxime bond.La thèse a porté sur l'étude de peptides et d'oligonucléotides par fragmentation dans la source électrospray d'un Quattro II (Micromass). L'instrument a d'abord été évalué en mode positif avec des peptides thioester modèles puis en mode négatif avec des oligodésoxynucléotides bromés dont la fragmentation de la base modifiée permet sa localisation dans la séquence. La fragmentation de peptides acétal et diol (aldéhyde hydraté) conduit à un même ion final cyclique. L'hydratation de peptides aldéhyde a été étudiée en solution par RMN et en phase gazeuse par fragmentation dans la source. Les informations issues de ce travail ont permis une meilleure compréhension du mécanisme de la réaction d'oximation

Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide

International audienceThe histidine-rich GLFHAIAHFIHGGWHGLIHGWYG peptide (H5WYG) coordinates a Zn2+ ion and forms a stable peptide-metal complex promoting membrane fusion at physiologic pH. In our previous article titled 'Histidine-rich peptide: evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH' in Journal of Mass Spectrometry (2009, 44,81-89), tandem mass spectrometry experiments have provided evidence for the binding of a single Zn2+ ion to H5WYG and suggested that this binding is shared between H-11, H-19 and probably H-15 residues. To clarify the involvement of these histidine residues in the binding to the Zn2+ ion and especially to remove the doubt about the implication of the H-15 residue, here we have used three H5WYG mutants termed H5WYGH11A, H5WYGH15A and H5WYGH19A, whose H-11, H-15 and H-19 residues were replaced with an alanine residue. The novelty introduced by these new tandem mass spectrometry experiments performed with the mutants is the demonstration that H-15 is involved in the binding of the single Zn2+ ion and that it may even favour the setting of another Zn2+ ion. Thus, the three histidines H-11, H-15 and H-19 could lead to a specific structuring of H5WYG that can promote membrane fusion upon the binding of zinc

Fragmentation dans une source à électronébulisation de biomolécules de synthèse (peptides thioester, acétal, aldéhyde et oligonucléotides bromés)

La thèse a porté sur l'étude de peptides et d'oligonucléotides par fragmentation dans la source électrospray d'un Quattro II (Micromass). L'instrument a d'abord été évalué en mode positif avec des peptides thioester modèles puis en mode négatif avec des oligodésoxynucléotides bromés dont la fragmentation de la base modifiée permet sa localisation dans la séquence.La fragmentation de peptides acétal et diol (aldéhyde hydraté) conduit à un même ion final cyclique. L'hydratation de peptides aldéhyde a été étudiée en solution par RMN et en phase gazeuse par fragmentation dans la source. Les informations issues de ce travail ont permis une meilleure compréhension du mécanisme de la réaction d'oximation.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF

Synthesis of peptide di-aldehyde precursor for stepwise chemoselective ligations via oxime bonds

International audienceTo synthezise a triple-function branched peptide in a modular way, we present a new strategy based on orthogonal generation of two aldehyde functions from an acetal and a 2-amino alcohol. Successive unmaskings of aldehyde functions of the stem peptide affords stepwise chemoselective ligations of two (aminooxy)acetyl peptides via oxime bonds

In-source fragmentation of peptide aldehydes and acetals: influence of peptide length and charge state

International audienceThe use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared

A single run LC-MS/MS method for phospholipidomics

International audienc

Characterization of glycosyl inositol phosphoryl ceramides from plants and fungi by mass spectrometry

SPE IPMInternational audienceAlthough glycosyl inositol phosphoryl ceramides (GIPCs) represent the most abundant class of sphingolipids in plants, they still remain poorly characterized in terms of structure and biodiversity. More than 50 years after their discovery, little is known about their subcellular distribution and their exact roles in membrane structure and biological functions. This review is focused on extraction and characterization methods of GIPCs occurring in plants and fungi. Global methods for characterizing ceramide moieties of GIPCs revealed the structures of long-chain bases (LCBs) and fatty acids (FAs): LCBs are dominated by tri-hydroxylated molecules such as monounsaturated and saturated phytosphingosine (t18:1 and t18:0, respectively) in plants and mainly phytosphingosine (t18:0 and t20:0) in fungi; FA are generally 14-26 carbon atoms long in plants and 16-26 carbon atoms long in fungi, these chains being often hydroxylated in position 2. Mass spectrometry plays a pivotal role in the assessment of GIPC diversity and the characterization of their structures. Indeed, it allowed to determine that the core structure of GIPC polar heads in plants is Hex(R1)-HexA-IPC, with R1 being a hydroxyl, an amine, or a N-acetylamine group, whereas the core structure in fungi is Man-IPC. Notably, information gained from tandem mass spectrometry spectra was most useful to describe the huge variety of structures encountered in plants and fungi and reveal GIPCs with yet uncharacterized polar head structures, such as hexose-inositol phosphoceramide in Chondracanthus acicularis and (hexuronic acid)(4)-inositol phosphoceramide and hexose-(hexuronic acid)(3)-inositol phosphoceramide in Ulva lactuca

Caveolae-mediated effects of TNF-alpha on human skeletal muscle cells

Chronic diseases are characterized by the production of pro-inflammatory cytokines such than TNF-alpha and are frequently correlated with muscle wasting conditions. Among the pleiotropic effects of TNF-alpha within the cell, its binding to TNFR1 receptor has been shown to activate sphingomyelinases leading to the production of ceramides. Sphingomyelinases and TNF receptor have been localized within caveolae which are specialized RAFT enriched in cholesterol and sphingolipids. Because of their inverted omega shape, maintained by the oligomerization of specialized proteins, caveolins and cavins, caveolae serve as membrane reservoir therefore providing mechanical protection to plasma membranes. Although sphingolipids metabolites, caveolins and TNF-alpha/TNFR1 have been shown to independently interfere with muscle physiology, no data have clearly demonstrated their concerted action on muscle cell regeneration. In this context, our study aimed at studying the molecular mechanisms induced by TNF-alpha at the level of caveolae in LHCN-M2 human muscle satellite cells. Here we showed that TNF-alpha-induced production of ROS and nSMase activation requires caveolin. More strikingly, we have demonstrated that TNF-alpha induces the formation of additional caveolae at the plasma membrane of myoblasts. Furthermore, TNF-alpha prevents myoblast fusion suggesting that inflammation could modulate caveolae organization/function and satellite cell function

Phospholipid Profiles of Oleaginous Pressed Cakes Using NMR and Gas Chromatography

Camelina, flaxseed, hemp, sesame, and walnut cakes were analyzed for their phospholipid (PL) content and composition using P-31 and H-1 nuclear magnetic resonance and gas chromatography. The data evidenced variations between the sources in terms of (1) total lipid content and PL concentration, camelina cake being the richest source of PLs, (2) PL composition, phosphatidylcholine being the most abundant phospholipid in sesame and hemp cakes, whereas phosphatidylinositol represented about 25% of the total PLs in most cakes, and (3) fatty acid composition of the PLs, camelina cake being the richest source of omega 3 polyunsaturated fatty acids. These data may be useful to diversify the PL sources available and to provide PL fractions with specific nutritional or functional properties