Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelo\"id 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

Enolases: Storage compounds in seeds? Evidence from a proteomic comparison of zygotic and somatic embryos of Cyclamen persicum Mill

Somatic embryogenesis is well established for the economic relevant ornamental crop Cyclamen and thus could supplement the elaborate propagation via seeds. However, the use of somatic embryogenesis for commercial large scale propagation is still limited due to physiological disorders and asynchronous development within emerged embryos. To overcome these problems, profound knowledge of the physiological processes in Cyclamen embryogenesis is essential. Thus, the proteomes of somatic and zygotic embryos were characterised in a comparative approach. Protein separation via two dimensional IEF-SDS PAGE led to a resolution of more than 1,000 protein spots/gel. Overall, 246 proteins were of differential abundance in the two tissues compared. Mass spectrometry analysis of the 300 most abundant protein spots resulted in the identification of 247 proteins, which represent 90 distinct protein species. Fifty-five percent of the 247 proteins belong to only three physiological categories: glycolysis, protein folding and stress response. The latter physiological process was especially predominant in the somatic embryos. Remarkably, the glycolytic enzyme enolase was the protein most frequently detected and thus is supposed to play an important role in Cyclamen embryogenesis. Data are presented that indicate involvement of "small enolases" as storage proteins in Cyclamen. A digital reference map was established via a novel software tool for the web-based presentation of proteome data linked to KEGG and ExPasy protein-databases and both were made publicly available online. © 2011 Springer Science+Business Media B.V

Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%–35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10−6% of total proteins, by weight. Signal peptide recovery ranged between 5%–100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction

Proteomic Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved in Vesicular Traffic and Nutritional Adaptation

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation

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

web publisher www.interscience.wiley.comInternational audienceThe 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

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.Comment: website publisher http://www.interscience.wiley.co

Early manganese-toxicity response in Vigna unguiculata L. - A proteomic and transcriptomic study

The apoplast is known to play a predominant role in the expression of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.) leaves. To unravel early Mn-toxicity responses after 1-3 days Mn treatment also in the leaf symplast, we studied the symplastic reactions induced by Mn in two cultivars differing in Mn tolerance on a total cellular level. Comparative proteome analyses of plants exposed to low or high Mn allowed to identify proteins specifically affected by Mn, particularly in the Mn-sensitive cowpea cultivar. These proteins are involved in CO2 fixation, stabilization of the Mn cluster of the photosystem II, pathogenesis-response reactions and protein degradation. Chloroplastic proteins important for CO2 fixation and photosynthesis were of lower abundance upon Mn stress suggesting scavenging of metabolic energy for a specific stress response. Transcriptome analyses supported these findings, but additionally revealed an upregulation of genes involved in signal transduction only in the Mn-sensitive cultivar. In conclusion, a coordinated interplay of apoplastic and symplastic reactions seems to be important during the Mn-stress response in cowpea. © 2008 Wiley-VCH Verlag GmbH &amp; Co. KGaA

Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses

The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents