High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore.

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple

'Palaeoshellomics' reveals the use of freshwater mother-of-pearl in prehistory

The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including 'palaeoshellomics', the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of 'double-buttons'. This craft is found throughout Europe between 4200-3800 BCE, highlighting the ornament-makers' profound knowledge of the biogeosphere and the existence of cross-cultural traditions

Apport de la protéomique à la médecine transfusionnelle (étude de l'impact des traitements d'inactivation des agents pathogènes et des conditions de stockage sur les protéines plasmatiques)

Bien que la protéomique ait été largement appliquée pour l étude du plasma humain, son application dans le domaine de la transfusion sanguine reste peu employée. En collaboration avec l EFS, l objectif de cette thèse a donc été de proposer des outils analytiques destinés à évaluer l impact des traitements d inactivation des agents pathogènes et des conditions de stockage sur les protéines plasmatiques. Le traitement au bleu de méthylène est le traitement d inactivation virale le plus utilisé en France. Une approche globale et ciblée se sont intéressées aux modifications induites par ce traitement photochimique. Plusieurs modifications, notamment sur les sous-unités du fibrinogène, ont pu être identifiées, après analyse nanoLC-nanoESI-Qh-FT-ICR MS. L origine de la diminution d activité du fibrinogène a pu ainsi être expliquée. Une étude thermique a permis d identifier un marqueur de dégradation plasmatique: la RBP4. Dans le plasma, elle forme un complexe avec la transthyrétine. Lors de la dégradation du plasma, ce complexe se dissocie. Une méthode de quantification absolue, basée sur des peptides AQUA, a été développée permettant de doser RPB4 libérée dans le plasma au cours de la conservation du plasma. Enfin, deux matrices innovantes pour l électrophorèse sur gel ont été évaluées pour la séparation de protéines plasmatiques. L une incorpore un polymère préformé, le dextran, à une solution d acrylamide classique. L autre fait appel à un polymère hydrophile, le NAT. Toutes deux présentent de bonnes propriétés optiques et mécaniques, augmentent significativement la résolution des spots protéiques et facilitent l identification des protéines par MS.Proteomics has been widely applied to study plasmatic proteins; its application to the field of transfusion medicine is s quite recent. In partnership with the French blood agency (EFS), the main objective of the Ph.D work was to provide analytical tools to evaluate the impact of pathogen inactivation treatments and storage conditions on plasmatic proteins. Photochemical treatment using methylene-blue is the most used for pathogen inactivation in France. Both a global and targeted studies were carried to determine the proteins modifications involved by this treatment. Based on nanoLC-nanoESI-Qh-FT-ICR MS analyses, several modifications were pointed out, especially targeting the sub-unit of fibrinogen. This allows the decrease in fibrinogen clottability after methylene-blue treatment to be explained.A study of thermal degradation on plasma sample pointed out a new marker of plasma degradation: the RBP4. It circulates associated to with transthyretin as a macromolecular complex: during degradation, this complex dissociates releasing RBP4 in plasma. An absolute quantification method was developed using AQUA peptides to assay the amount of the free form of RBP4 in plasma during storage.Two innovative matrices for gel electrophoresis were developed and evaluated for plasma protein separation. One of them relies on the use on a preformed polymer incorporated prior to acrylamide polymerization. The other one is based on a hydroxylated acrylamide monomer, the N-acryol-tris(hydroxymethyl)-amino methane. Both exhibited interesting optical and mechanical properties, enhanced spot resolution and outstanding protein/peptide recovery, which facilitates protein identification by MS.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Étude des modifications des protéines induites par la fumée de cigarette

La fumée de cigarette, reconnue comme étant carcinogène, est un aérosol complexe composé de plusieurs milliers de substances chimiques susceptibles de réagir avec les biomolécules. Aussi l identification des modifications post-traductionnelles des protéines induites par la fumée de cigarette s avère être un enjeu majeur à la compréhension de certaines pathologies telles que les cancers ou les maladies cardiovasculaires.L objectif de ce travail a été de développer une méthode d analyse basée sur la spectrométrie de masse à haute résolution afin d identifier et de localiser les modifications des protéines exposées à la phase gazeuse de la fumée de cigarette. Deux techniques ont été utilisées : l analyse MALDI-TOF-TOF et l analyse nanoESI-FT-ICR couplée à la nano chromatographie liquide. Dans le but d optimiser la réaction avec la fumée de cigarette, des modèles de réactions ont été développés sur des peptides et protéines standards. Ainsi des réactions d oxydation et de nitration ont été effectués à partir des radicaux libres HO et NO2 tandis que des réactions de formation d adduits ont été réalisées à partir d un équivalent d aldéhyde (formaldéhyde, acétaldehyde, malonaldéhyde, acroléine, crotonaldéhyde et glyoxal). Les mêmes standards protéiques ont ensuite été exposés à la fumée de cigarette à l aide d une machine à fumer. Par comparaison avec les modèles de réaction, des adduits provenant de l acétaldéhyde, de l acroléine et du crotonaldéhyde ont pu être identifiés et localisés sur des résidus lysines spécifiques. Ces résultats ont permis de mettre en évidence que les protéines étaient majoritairement modifiées par les aldéhydes contenus dans la fumée de cigarette.Cigarette smoke, well known to be carcinogen, is a high complex aerosol composed of further thousands of chemicals able to react with biomolecules. Also the identification of post-translational protein modifications induced by cigarette smoke is a major stake for the understanding of some pathology like cancer and cardiovascular disease. The aim of this work was to develop an analytical method based on high resolution mass spectrometry in order to identify and localize protein modifications induced by cigarette smoke. Two techniques were used: a MALDI-TOF-TOF analysis and a on line nano liquid chromatography nanoESI-FT-ICR analysis. In order to optimize reaction with cigarette smoke, model reactions were developed on standard peptides and protein. Thus oxidation and nitration were realized using HO and NO2 free radicals while formation of adducts was performed using 1 eq of aldehydes (formaldehyde, acetaldehyde, malonaldehyde, acrolein, crotonaldehyde and glyoxal). Same proteins were then exposed to cigarette smoke using a smoking machine.By comparison with model reactions, adduct coming from acetaldehyde, acrolein and crotonaldehyde were identified and localized on specific lysine amino acids. These results allowed highlighting that proteins are principally modified by aldehydes contained in cigarette smoke.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Biophysical Chemistry

Little is known about structural alterations of proteins within the polymeric films of paints. For the first time, hydrogen‑deuterium exchange mass spectrometry (HDX-MS) was implemented to explore the conformational alterations of proteins resulting from their interaction with inorganic pigments within the early stages of the paint film formation. Intact protein analysis and bottom-up electrospray-ionisation mass spectrometry strategies combined with progressively increasing deuterium incubation times were used to compare the protein structures of the model protein hen egg-white lysozyme (HEWL) extracted from newly dried non-pigmented films and newly dried films made from a freshly made mixture of HEWL with lead white pigment (2PbCO3 Pb(OH)2). The action of other pigments was also investigated, expanding the HDX study with a global approach to paint models of HEWL mixed with zinc white (ZnO), cinnabar (HgS) and red lead (Pb3O4) pigments. The results show structural modifications of HEWL induced by the interaction with the pigment metal ions during the paint formulation after drying and prior to ageing. Both the charge distribution of HEWL proteoforms, its oxidation rate and its deuterium absorption rate, were influenced by the pigment type, providing the first insights into the correlation of pigment type/metal cation to specific chemistries related to protein stability.Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-60 ITN

Nouvelle génération de colonnes capillaires ouvertes de dimension micronique pour l'analyse protéomique basées sur la fonctionnalisation de surface

La miniaturisation des systèmes séparatifs est en plein développement aujourd hui. Cette évolution se justifie par le très faible volume de l échantillon et la nécessité croissante de gagner du temps d analyse. Cette thèse comporte deux parties. La première partie consiste à développer une nouvelle génération de colonnes capillaires ouvertes ( open tubular ) de dimension micronique basées sur la fonctionnalisation de surface et de caractériser leurs propriétés en analyse protéomique pour la séparation de digests peptidiques par chromatographie liquide en phase inverse. La fonctionnalisation de surface a été réalisée après activation de la paroi de silice du capillaire par greffage de dérivés silylés comprenant le motif méthacrylate de glycidyle. La fonction époxyde a ensuite été ouverte par des amines primaires aliphatiques afin d augmenter l hydrophobicité de la phase stationnaire. Cette stratégie en deux étapes a l avantage de permettre de dissocier le greffage de l introduction du motif fonctionnel et de permettre une plus grande variété de fonctionnalisation. Ces colonnes ont été testées sur des peptides standards ou sur des digests peptidiques avec une analyse par spectrométrie de masse MALDI-TOF hors-ligne et en ligne en couplage nanoLC nanoESI-trappe ionique. Les effets des différents paramètres sur la rétention des peptides ont été étudiés en particulier la longueur de la chaîne alkyle. Les tests montrent que chaque fois que l on diminue la longueur de la chaîne carbonée de l amine, les peptides sont élués à un plus faible pourcentage d acétonitrile. En effet le raccourcissement de la chaîne carbonée conduit à la décroissance des interactions hydrophobes, ce qui conduit les peptides à être élués plus rapidement. La deuxième partie de la thèse présente les premiers développements de greffage de polymère à empreinte moléculaire (acronyme anglais MIP pour Molecular Imprinted Polymer) pour la séparation de peptides et de protéines. La synthèse d une phase à empreinte moléculaire est réalisée à partir d un mélange de monomères portant des fonctions acides ou basiques et d une molécule empreinte qui interagit au cours de la polymérisation par des liaisons non covalentes. Une fois la polymérisation achevée, la molécule empreinte est éliminée de la matrice polymérique, ce qui conduit à la formation d un polymère rigide renfermant des sites de reconnaissance spécifiques de la molécule modèle. Les colonnes à empreinte moléculaire visent à retenir sélectivement la molécule ciblée ou des molécules proches en taille et en structure, au sein d un mélange complexe par des interactions spécifiques. Une affinité entre deux peptides différents a été obtenue avec une bonne sélectivité. La préparation des colonnes MIP et NIP (polymère non imprimé) a été effectuée afin de comparer le pouvoir de rétention des deux matériaux et de vérifier la sélectivité de recapture du MIP par rapport au NIP vis-à-vis de la molécule cible. Ces colonnes ont également été testées par spectrométrie de masse de type MALDI-TOF.The miniaturization of separative systems is continuously developing today. This emergence is due to the very low sample volume and the increasing need to save analysis time. The present thesis comprises two parts. The first one consists on developing a new generation of open capillary columns ("open tubular") based on micron-scale surface functionalization and characterization of their properties in proteomics analysis for the separation of peptide digests by reversed-phase liquid chromatography. The surface functionalization was carried out after activating silica capillary wall by grafting silylated derivatives including glycidyl methacrylate. The epoxide function was then broken by aliphatic primary amines to increase the hydrophobicity of the stationary phase. This two-step strategy has the advantage of allowing a split grafting of the functional unit and a wider variety of functionalizations. These columns were tested on standard peptides or peptide digests with analysis by MALDI-TOF offline and online nanoLC nanoESI-coupled ion trap. The effects of various parameters, particularly the length of the alkyl chain, on the retention of peptides were studied. The obtained results show that by decreasing the length of the amine carbon chain, peptides are eluted at a lower percentage of acetonitrile. In fact the shortening of the carbon chain results in the decrease of hydrophobic interactions and in faster peptides elution.The second part of this thesis presents preliminary developments of polymer grafting of type MIP (Molecular Imprinted Polymers) for the separation of peptides and proteins. The synthesis of molecularly imprinted phase is made from a mixture of monomers having acid or basic functions and imprint molecules that interact during the polymerization by non-covalent bonds. Once the polymerization is complete, the imprint molecules are removed from the polymer matrix, which results in the formation of a rigid polymer containing recognition sites that are specific to the model molecules. The molecularly imprinted columns were designed to selectively retain the target molecules or molecules similar in size and in structure, within a complex mixture by specific interactions. Affinity between two different peptides was obtained with good selectivity. Preparation of MIP and NIP (No Imprinted Polymers) columns was performed to compare the retention capacity of both materials and to verify the selectivity of the MIP uptake compared to NIP with respect to the target molecule. These columns were also tested by mass spectrometry MALDI-TOF.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Characterization of Isomers of Lipid A from Pseudomonas aeruginosa PAO1 by Liquid Chromatography with Tandem Mass Spectrometry with Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation

Lipopolysaccharides (LPS) constitute the outermost layer of Gram-negative bacteria and consequently play an important role in bacterial infections. In order to address public health issues posed by Gram-negative bacteria, it is necessary to elucidate the structure of the molecular actors at the forefront of infections. LPS virulence and toxicity are partially modulated by lipid A, a hydrophobic saccharolipid that anchors LPS to the bacterial outer membrane. Understanding the lipid A structure is inherently intertwined with understanding its role as an endotoxin. Accordingly, several successful strategies incorporating tandem mass spectrometry have been applied toward the structural analysis of lipid A. Herein, a shotgun HCD strategy was applied toward the characterization of the lipid A profile of Pseudomonas aeruginosa PAO1. This analysis was enhanced by the development of an LC-MS/MS approach to eliminate isomeric signals in the MS/MS spectra that confounded characterization. Importantly, combining reverse phase chromatography with HCD and ultraviolet photodissociation analyses of the lipid A profile revealed the presence of previously unreported lipid A acyl chain positional isomers. Altogether, these strategies provide the most in-depth structural and molecular characterization of PAO1 lipid A to date

Analysis of the Phospholipid Profile of the Collection Strain PAO1 and Clinical Isolates of Pseudomonas aeruginosa in Relation to Their Attachment Capacity

Bacteria form multicellular and resistant structures named biofilms. Biofilm formation starts with the attachment phase, and the molecular actors involved in this phase, except adhesins, are poorly characterized. There is growing evidence that phospholipids are more than simple structural bricks. They are involved in bacterial adaptive physiology, but little is known about their role in biofilm formation. Here, we report a mass spectrometry analysis of the phospholipid (PL) profile of several strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients. The aim of our study was to evaluate a possible link between the PL profile of a strain and its attachment phenotype. Our results showed that PL profile is strongly strain-dependent. The PL profile of P. aeruginosa PAO1, a collection strain, was different from those of 10 clinical isolates characterized either by a very low or a very high attachment capacity. We observed also that the clinical strain's PL profiles varied even more importantly between isolates. By comparing groups of strains having similar attachment capacities, we identified one PL, PE 18:1-18:1, as a potential molecular actor involved in attachment, the first step in biofilm formation. This PL represents a possible target in the fight against biofilms

Comparative Proteomic and Transcriptomic Analysis of Follistatin-Induced Skeletal Muscle Hypertrophy.

Skeletal muscle, the most abundant body tissue, plays vital roles in locomotion and metabolism. Myostatin is a negative regulator of skeletal muscle mass. In addition to increasing muscle mass, Myostatin inhibition impacts muscle contractility and energy metabolism. To decipher the mechanisms of action of the Myostatin inhibitors, we used proteomic and transcriptomic approaches to investigate the changes induced in skeletal muscles of transgenic mice overexpressing Follistatin, a physiological Myostatin inhibitor. Our proteomic workflow included a fractionation step to identify weakly expressed proteins and a comparison of fast versus slow muscles. Functional annotation of altered proteins supports the phenotypic changes induced by Myostatin inhibition, including modifications in energy metabolism, fiber type, insulin and calcium signaling, as well as membrane repair and regeneration. Less than 10% of the differentially expressed proteins were found to be also regulated at the mRNA level but the Biological Process annotation, and the KEGG pathways analysis of transcriptomic results shows a great concordance with the proteomic data. Thus this study describes the most extensive omics analysis of muscle overexpressing Follistatin, providing molecular-level insights to explain the observed muscle phenotypic changes