Apport de la spectrométrie de masse à l'étude de modifications post-traductionnelles et à la protéomique

Ces 10 dernières années ont vu une évolution rapide de la spectrométrie de masse en fonction des besoins croissant des biologistes, dès l'avènement des nouvelles techniques d'ionisation dite douces que sont le MALDI (Désorption Ionisation laser assisté par matrice) et l'ESI (ionisation electrospray) permettant enfin la détection intacte de molécules d'origine biologique.Ce travail de thèse a porté principalement sur le choix et la mise au point de la stratégie instrumentale et analytique pour mener à bien quatre collaborations, soient quatre problèmes montrant à la fois la diversité des approches et la multiplicité des solutions apportées par la spectrométrie de masse. Le premier sujet a porté sur l'isolement de peptides biologiquement actifs dans l'hémolymphe de drosophiles immunisées. Nous avons réalisé le séquençage de novo par spectrométrie de masse de l'un de ces peptides bloqué en N-terminal, appelé DIM 13 (Drosophila Immune Induced molecule 13). Le second sujet a porté sur l'étude de la conformité structurale d'une enzyme, la dipeptidyl-diamino-peptidase. Nous avons pu établir la conformité de la protéine recombinante par rapport à la protéine naturelle et donné la description détaillée de ses modifications post-traductionnnelles.Le troisième sujet a consisté à l'établissement du protéome de la rétine de souris. Une première approche a conduit à l'identification d'une centaine de protéines de la rétine. Puis dans une seconde approche une étude différentielle a été entreprise comparant les protéines observées sur gel SDS PAGE 2D obtenu à partir de rétines de souris normales versus les protéines obtenues à partir de rétines de souris rd1 (retinal degenerescence 1). Enfin la dernière étude a porté sur la description de la composition en protéines d'un complexe de facteur de transcription, le complexe TFTC. Notre approche a permis d'ajouter 2 nouvelles protéines à la liste des protéine déjà identifiées par des méthodes de biochimie classique.Last past decade has shown a rapid evolution of mass spectrometry with the increasing demands of biologists since the birth of new soft ionisation techniques such MALDI (Matrix Assisted Laser Desorption Ionisation) and ESI (Electrospray Ionisation), allowing the detection of undazzled biological molecules.This thesis work describes the choice and the development of the instrumental and analytical strategy to carry out four different collaborations, showing by the way the diversity of the approaches and the multiplicity of mass spectrometry solutions.The first topic concerned isolation in drosophila hemolymph of biologically active peptides. We realized the de novo sequencing by Ion Trap mass spectrometry of an N terminally blocked peptide named DIM 13 (Drosophila Immune Induced Molecule 13).The second topic aimed the structural study of an recombinant enzyme, the dipeptidyl-diaminopeptidase. We established the conformity of the primary structure of the recombinant protein versus the natural one and gave a detailed description of its post-translationnal modifications.The third topic concerned the establishment of the proteome of mouse retina. A first approach led to the identification by peptide mass fingerprinting and databank search of one hundred retina proteins. In a second approach we compared proteins identified from normal mouse retina versus rd1 retina (retinal degenerescence 1).The last study gave the description of proteins from a transcriptional factor, TFTC. Our approach permitted the identification of two additional proteins, from those already identified by more classical methods.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Novel subunits of the TATA binding protein free TAFII-containing transcription complex identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry following one-dimensional gel electrophoresis

International audienceInitiation of transcription of protein-encoding genes by RNA polymerase II was thought to require the transcription factor II D (TF(II)D), a complex comprising the TATA binding protein (TBP) and TBP-associated factors. However, another multiprotein complex isolated more recently and called TFTC (TBP-free TAF(II )containing complex), was shown to mediate initiation of RNA polymerase II (Pol II) transcription in the absence of TF(II)D as well as specific acetylation of histone H3 in a nucleosomal context. Several subunits of the TFTC complex were already identified using classical methods such as Edman based microsequencing and Western blot analysis. In this article we present a mass spectrometry based proteomic approach to confirm previous results and to identify other possible subunits of the TFTC complex. The TFTC complex was separated on one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis and analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting. Identifications were realized after databank searches. This new characterization of TFTC complex confirmed the presence of already described subunits (TRRAP, GCN5, SAP130/KIA0017, TAF(II)150, TAF(II)135, TAF(II)100, TAF(II)80, TAF(II)20, SPT3 and PAF65beta). Moreover, a good coverage of these sequences was obtained. Interestingly, TAF(II)32 and PAF6alpha were also determined as potential novel subunits of TFTC. These results together show the suitability and the great potential of this method and offer new perspectives in fundamental studies of transcription factor complexes

Differential proteomic analysis of the mouse retina: the induction of crystallin proteins by retinal degeneration in the rd1 mouse

International audienceWe have applied proteomic analysis to the degeneration of photoreceptors. In the rd1 mouse, a recessive mutation in the PDE6B gene leads to rapid loss of rods through apoptosis. By 5 wk postnatal, virtually all rod photoreceptors have degenerated, leaving one row of cones that degenerates secondarily. In order to assess comparative protein expression, proteins extracted from whole retina were resolved on a two-dimensional gel and identified by mass spectrometry combined with database screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry coupled to peptide mass fingerprinting was sufficient to identify most of the proteins, the remaining being identified with additional sequence information obtained by nano-electrospray ionization tandem mass spectrometry or liquid chromatography tandem mass spectrometry. The study revealed 212 spots, grouped into 109 different proteins. Differential analysis showed loss of proteins involved in the rod-specific phototransduction cascade, as well as induction of proteins from the crystallin family, in response to retinal degeneration. Identification of such pathways may contribute to new therapeutic approaches

Transglutaminase-3 Enzyme: A Putative Actor in Human Hair Shaft Scaffolding?

The family of transglutaminases (TGase) is known to be involved in terminal differentiation processes in the epidermis. These enzymes contribute also to the physical resistance and the preservation of the hair follicle structure. Our particular interest in hair fiber keratinization led us to focus on the TGase 3, exclusively expressed in the hair shaft. To date its function is still to be elucidated, thus we have developed a multidisciplinary approach in order to define the localization, activity, and substrates of TGase 3. The hair fiber is characterized by the expression of specific proteins essentially consisting of keratin intermediate filaments and keratin-associated proteins (KAPs), which are essential for the formation of a rigid hair shaft through their extensive disulfide cross-links. Gel electrophoresis combined with mass spectrometry experiments revealed an unexpected protein migration pattern, suggesting the existence of covalent interactions other than disulfide bonds. Western blot and amino-acid analysis revealed the presence of γ-glutamyl-ε-lysine isopeptide linkages that could constitute this second covalent network. Our hypothesis is that TGase 3-driven specific isopeptide bonds between intermediate filaments and KAPs participate to the progressive scaffolding of the hair shaft

: RdCVF

International audienceRetinitis pigmentosa is an untreatable, inherited retinal disease that leads to blindness. The disease initiates with the loss of night vision due to rod photoreceptor degeneration, followed by irreversible, progressive loss of cone photoreceptor. Cone loss is responsible for the main visual handicap, as cones are essential for day and high-acuity vision. Their loss is indirect, as most genes associated with retinitis pigmentosa are not expressed by these cells. We previously showed that factors secreted from rods are essential for cone viability. Here we identified one such trophic factor by expression cloning and named it rod-derived cone viability factor (RdCVF). RdCVF is a truncated thioredoxin-like protein specifically expressed by photoreceptors. The identification of this protein offers new treatment possibilities for retinitis pigmentosa

Multi-omics analysis to decipher the molecular link between chronic exposure to pollution and human skin dysfunction

International audienceEnvironmental pollution is composed of several factors, namely particulate matter (PM2.5, PM10), ozone and Ultra Violet (UV) rays among others and first and the most exposed tissue to these substances is the skin epidermis. It has been established that several skin disorders such as eczema, acne, lentigines and wrinkles are aggravated by exposure to atmospheric pollution. While pollutants can interact with skin surface, contamination of deep skin by ultrafine particles or Polycyclic aromatic hydrocarbons (PAH) might be explained by their presence in blood and hair cortex. Molecular mechanisms leading to skin dysfunction due to pollution exposure have been poorly explored in humans. In addition to various host skin components, cutaneous microbiome is another target of these environment aggressors and can actively contribute to visible clinical manifestation such as wrinkles and aging. The present study aimed to investigate the association between pollution exposure, skin microbiota, metabolites and skin clinical signs in women from two cities with different pollution levels. Untargeted metabolomics and targeted proteins were analyzed from D-Squame samples from healthy women (n = 67 per city), aged 25–45 years and living for at least 15 years in the Chinese cities of Baoding (used as a model of polluted area) and Dalian (control area with lower level of pollution). Additional samples by swabs were collected from the cheeks from the same population and microbiome was analysed using bacterial 16S rRNA as well as fungal ITS1 amplicon sequencing and metagenomics analysis. The level of exposure to pollution was assessed individually by the analysis of polycyclic aromatic hydrocarbons (PAH) and their metabolites in hair samples collected from each participant. All the participants of the study were assessed for the skin clinical parameters (acne, wrinkles, pigmented spots etc.). Women from the two cities (polluted and less polluted) showed distinct metabolic profiles and alterations in skin microbiome. Profiling data from 350 identified metabolites, 143 microbes and 39 PAH served to characterize biochemical events that correlate with pollution exposure. Finally, using multiblock data analysis methods, we obtained a potential molecular map consisting of multi-omics signatures that correlated with the presence of skin pigmentation dysfunction in individuals living in a polluted environment. Overall, these signatures point towards macromolecular alterations by pollution that could manifest as clinical sign of early skin pigmentation and/or other imperfections