Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis

Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry

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 macrophagesComment: Nanosafe2010: International Conference on Safe Production and Use of Nanomaterials 16-18 November 2010, Grenoble, France, Grenoble : France (2010

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.This work was supported by Regione Autonoma della Sardegna (http://www.regione.sardegna.it/)Pubblicat

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

Detergents and chaotropes for protein solubilization before two-dimensional electrophoresis

Because of the outstanding ability of two-dimensional electrophoresis to separate complex mixtures of intact proteins, it would be advantageous to apply it to all types of proteins, including hydrophobic and membrane proteins. Unfortunately, poor solubility hampers the analysis of these molecules. As these problems arise mainly in the extraction and isoelectric focusing steps, the solution is to improve protein solubility under the conditions prevailing during isoelectric focusing. This chapter describes the use of chaotropes and novel detergents to enhance protein solubility during sample extraction and isoelectric focussing, and discusses the contribution of these compounds to improving proteomic analysis of membrane proteins

Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery

Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence

The Whereabouts of 2D Gels in Quantitative Proteomics

Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using 2D gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of 2D gels is performed

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress

Solubilization of Proteins in 2DE: An Outline

Protein solubilization for two-dimensional electrophoresis (2DE) has to break molecular interactions to separate the biological contents of the material of interest into isolated and intact polypeptides. This must be carried out in conditions compatible with the first dimension of 2DE, namely isoelectric focusing. In addition, the extraction process must enable easy removal of any nonprotein component interfering with the isoelectric focusing. The constraints brought in this process by the peculiar features of isoelectric focusing are discussed, as well as their consequences in terms of possible solutions and limits for the solubilization process