Thierry Rabilloud

Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis

The solubilizing power of various nonionic and zwitterionic detergents as membrane protein solubilizers for two‐dimensional electrophoresis was investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf membrane proteins were used as model systems. Efficient detergents could be found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar heads. Among the commercially available nonionic detergents, dodecyl maltoside and decaethylene glycol mono hexadecyl ether proved most efficient. They complement the more classical sulfobetaine detergents to widen the scope of useful detergents for the solubilization of membrane proteins in proteomics.

A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress.

Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells.

A survey of the plant mitochondrial proteome in relation to development.

To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two‐dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two‐dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix‐assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post‐translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.

Membrane proteins ride shotgun

A new mass spectrometry–based approach identifies and characterizes membrane proteins on a large scale.

About the mechanism of interference of silver staining with peptide mass spectrometry

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde‐free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.

Progress in the definition of a reference human mitochondrial proteome

Owing to the complexity of higher eukaryotic cells, a complete proteome is likely to be very difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and “physiology” of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting. The optimization steps in two‐dimensional electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.

About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis

The influence of thiol blocking on the resolution of basic proteins by two‐dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in‐gel rehydration on wide gradients (e.g. 3–10 and 4–12), but anodic cup‐loading was still required for basic gradients (e.g. 6–12 or 8–12). These results demonstrate that thiol‐related problems are not solely responsible for streaking of basic proteins on two‐dimensional gels.

High Expression of Antioxidant Proteins in Dendritic Cells Possible Implications in Atherosclerosis

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H2O2, is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor κB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor κB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Concepts and therapeutic perspectives of proteomics

The present paper aims at clarifying some important aspects of proteomics, i.e. the large scale analysis of proteins. To this purpose, the main types of proteomic analyses are presented, i.e. those aiming at determining expression levels and those aiming at unravelling protein-protein interactions networks. Their performances and limitations are outlined, as well as their potential applications in biomedicine, to give an reasoned view of the current state of the art.

L’analyse protéomique : concepts, réalités et perspectives en thérapeutique

587 Alors que l’essentiel de la recherche en biologie avait suivi le chemin réductionniste et hypothético-déductif, les dernières années ont été marquées par l’émergence du concept de biologie à grande échelle. Ce concept repose sur le postulat selon lequel les systèmes biologiques fonctionnent comme des réseaux complexes, et qu’une étude à l’échelle la plus grande possible peut apporter un surcroît de connaissances. La conceptualisation de cette approche, qui remonte au début des années 80 [1], a conduit à l’application de cette démarche aux macromolécules biologiques. Du fait de la maturité des outils utilisés pour le clonage et le séquençage de l’ADN, cette molécule a été étudiée la première à l’échelle de génomes entiers, et les progrès ont été impressionnants depuis la première publication de la séquence complète d’un génome [2]. En revanche, si le génome fournit le répertoire de chaque cellule d’un organisme, la réalité cellulaire va dépendre de l’emploi de ce répertoire en termes de choix des gènes exprimés, de niveaux d’expression des produits géniques, et de modulation de la fonctionnalité des protéines, par exemple par des modifications post-traductionnelles. De plus, il apparaît de plus en plus clairement que les protéines ne fonctionnent pas dans la cellule de façon isolée, mais sous forme de complexes multimoléculaires. Ainsi, il devient évident que l’étude des protéines sera plus proche de la physiologie cellulaire. Une branche nouvelle de la biochimie visant à l’étude à grande échelle des protéines s’est donc développée récemment. De même que l’ensemble des gènes d’un organisme forme son génome, l’ensemble des protéines d’une cellule ou d’un tissu forme son protéome, et cette discipline a reçu le nom d’analyse protéomique. Cette discipline se subdivise en deux tendances selon la propriété des protéines qui est examinée en priorité, et l’on distingue ainsi la protéomique d’expression et la protéomique d’interactions.