arXiv subjects

Sylvie Luche

Publications and source records attributed to Sylvie Luche.

At least 19 recordsLinked to original sources

arXiv2018

Zinc oxide induces the stringent response and major reorientations in the central metabolism of Bacillus subtilis

Microorganisms, such as bacteria, are one of the first targets of nanoparticles in the environment. In this study, we tested the effect of two nanoparticles, ZnO and TiO2, with the salt ZnSO4 as the control, on the Gram-positive bacterium Bacillus subtilis by 2D gel electrophoresis-based proteomics. Despite a significant effect on viability (LD50), TiO2 NPs had no detectable effect on the proteomic pattern, while ZnO NPs and ZnSO4 significantly modified B. subtilis metabolism. These results allowed us to conclude that the effects of ZnO observed in this work were mainly attributable to Zn dissolution in the culture media. Proteomic analysis highlighted twelve modulated proteins related to central metabolism: MetE and MccB (cysteine metabolism), OdhA, AspB, IolD, AnsB, PdhB and YtsJ (Krebs cycle) and XylA, YqjI, Drm and Tal (pentose phosphate pathway). Biochemical assays, such as free sulfhydryl, CoA-SH and malate dehydrogenase assays corroborated the observed central metabolism reorientation and showed that Zn stress induced oxidative stress, probably as a consequence of thiol chelation stress by Zn ions. The other patterns affected by ZnO and ZnSO4 were the stringent response and the general stress response. Nine proteins involved in or controlled by the stringent response showed a modified expression profile in the presence of ZnO NPs or ZnSO4: YwaC, SigH, YtxH, YtzB, TufA, RplJ, RpsB, PdhB and Mbl. An increase in the ppGpp concentration confirmed the involvement of the stringent response during a Zn stress. All these metabolic reorientations in response to Zn stress were probably the result of complex regulatory mechanisms including at least the stringent response via YwaC.

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arXiv2012

Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloïd cell lines nuclear proteomes

One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta 2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of observed changes are due to post-translational modifications, thereby a exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications.

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arXiv2012

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.

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arXiv2011

Effects of nanoparticles on murine macrophages

Metallic nanoparticles are more and more widely used in an increasing number of applications. Consequently, they are more and more present in the environment, and the risk that they may represent for human health must be evaluated. This requires to increase our knowledge of the cellular responses to nanoparticles. In this context, macrophages appear as an attractive system. They play a major role in eliminating foreign matter, e.g. pathogens or infectious agents, by phagocytosis and inflammatory responses, and are thus highly likely to react to nanoparticles. We have decided to study their responses to nanoparticles by a combination of classical and wide-scope approaches such as proteomics. The long term goal of this study is the better understanding of the responses of macrophages to nanoparticles, and thus to help to assess their possible impact on human health. We chose as a model system bone marrow-derived macrophages and studied the effect of commonly used nanoparticles such as TiO2 and Cu. Classical responses of macrophage were characterized and proteomic approaches based on 2D gels of whole cell extracts were used. Preliminary proteomic data resulting from whole cell extracts showed different effects for TiO2-NPs and Cu-NPs. Modifications of the expression of several proteins involved in different pathways such as, for example, signal transduction, endosome-lysosome pathway, Krebs cycle, oxidative stress response have been underscored. These first results validate our proteomics approach and open a new wide field of investigation for NPs impact on macrophages

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arXiv2011

Improvements and simplifications in in-gel fluorescent detection of proteins using ruthenium II tris-(bathophenanthroline disulfonate): the poor man's fluorescent detection method

Fluorescent detection of proteins is a popular method of detection allying sensitivity, linearity and compatibility with mass spectrometry. Among the numerous methods described in the literature, staining with ruthenium II tris(bathophenanthroline disulfonate) is particularly cost-effective, but slightly cumbersome owing to difficulties in the preparation of the complex and complexity of staining protocols. We describe here the modifications on both aspects that allow to perform a higher contrast staining and offer a more robust method of complex preparation, thereby maximizing the advantages of the method.

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arXiv2010

Two-dimensional gel electrophoresis in proteomics: past, present and future

Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.

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arXiv2009

Silver Staining of Proteins in 2DE Gels

Silver staining detects proteins after electrophoretic separation on polyacrylamide gels. Its main positive features are its excellent sensitivity (in the low nanogram range) and the use of very simple and cheap equipment and chemicals. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation, and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 day after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.

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arXiv2009

Organelle proteomics

This unit describes strategies for studying the proteomes of organelles, which is one example of targeted proteomics. It relies heavily on previously published units dealing with organelle preparation, protein solubilization, and proteomics techniques. A specific commentary for organelle proteomics is provided. Specific protocols for the isolation of nuclei from various sources (cell cultures, tissues) are also provided.

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arXiv2008

Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update

The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field.

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arXiv2008

Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry

Protein detection methods after electrophoresis have to be sensitive, homogeneous, and not to impair downstream analysis of proteins by MS. Speed, low cost, and user friendliness are also favored features. Silver staining combines many of these features, but its compatibility with MS is limited. We describe here, a new variant of silver staining that is completely formaldehyde-free. Reducing sugars in alkaline borate buffer are used as developers. While keeping the benefits of silver staining, this method is shown to afford a much better performance in terms of compatibility with MS, both in PMF by MALDI and in LC/ESI/MS/MS.

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arXiv2008

Oxidative stress response: a proteomic view

The oxidative stress response is characterized by various effects on a range of biologic molecules. When examined at the protein level, both expression levels and protein modifications are altered by oxidative stress. While these effects have been studied in the past by classic biochemical methods, the recent onset of proteomics methods has allowed the oxidative stress response to be studied on a much wider scale. The input of proteomics in the study of oxidative stress response and in the evidence of an oxidative stress component in biologic phenomena is reviewed in this paper.

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arXiv2007

Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

Protein detection on SDS gels or on 2-D gels must combine several features, such as sensitivity, homogeneity from one protein to another, speed, low cost, and user-friendliness. For some applications, it is also interesting to have a nonfixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that coelectrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal CBB and the one of fluroescent ruthenium complexes. Detection is achieved within 1 h after the end of the electrophoretic process and does not use any fixing or toxic agent. The fluorescent SDS-carbocyanine-protein complexes can be detected either with a laser scanner with an excitation wavelength of 488 nm or with a UV table operating at 302 nm. Excellent sequence coverage in subsequent MS analysis of proteolytic peptides is also achieved with this detection method.

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arXiv2007

Silver staining of proteins in polyacrylamide gels

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.

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arXiv2006

A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins

A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300 000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above M(r) range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pK(a), as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6-200 kDa M(r) range, with minimal difficulties in the post electrophoretic identification processes.

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arXiv2006

Detergents and Chaotropes for Protein Solubilization before Two-Dimensional Electrophoresis

Because of the outstanding separating capabilities of two-dimensional electrophoresis for complete proteins, it would be advantageous to be able to apply it to all types of proteins. Unfortunately, severe solubility problems hamper the analysis of many classes of proteins, but especially membrane proteins. These problems arise mainly in the extraction and isoelectric focusing steps, and solutions are sought to improve protein solubility under the conditions prevailing during isoelectric focusing. These solutions deal mainly with chaotropes and new detergents, which are both able to enhance protein solubility. The input of these compounds in proteomics analysis of membrane proteins is discussed, as well as future directions.

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arXiv2006

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.

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arXiv2006

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.

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arXiv2006

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.

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