A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study

Albaum S, Hahne H, Otto A, et al. A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study. Proteome Science. 2011;9(1): 30.Background: Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up- or down-regulated proteins at a distinct time point but instead compare developmental stages of an organism or varying experimental conditions. Results: Although the scientific questions behind these experiments are of course manifold, there are, nevertheless, two questions that commonly arise: 1) which proteins are differentially regulated regarding the selected experimental conditions, and 2) are there groups of proteins that show similar abundance ratios, indicating that they have a similar turnover? We give advice on how these two questions can be answered and comprehensively compare a variety of commonly applied computational methods and their outcomes. Conclusions: This work provides guidance through the jungle of computational methods to analyze mass spectrometry-based isotope-labeled datasets and recommends an effective and easy-to-use evaluation strategy. We demonstrate our approach with three recently published datasets on Bacillus subtilis [1,2] and Corynebacterium glutamicum [3]. Special focus is placed on the application and validation of cluster analysis methods. All applied methods were implemented within the rich internet application QuPE [4]. Results can be found at http://qupe.cebitec.uni-bielefeld.de webcite

Bioleaching of Pyrite by Iron-Oxidizing Acidophiles under the Influence of Reactive Oxygen Species

After 24h of exposure to acidic media, pyrite generates reactive oxygen species (ROS). Freshly-crushed pyrite with grain sizes between 50-100 μm at a 5 % (w/v), pulp density generated 0.17 ± 0.01 mM H2O2, while 10% pyrite generated 0.29 ± 0.01 mM and 30 % pyrite generated approximately 0.83 ± 0.06 mM. These levels of H2O2 inhibit iron oxidation in iron-grown cells of AcidithiobacillusferrooxidansT but not in pyrite-grown cells. ROS originating from pyrite, which was incubated for 24 h in acidic medium, prohibited pyrite dissolution by iron-grown cells, while pyrite-grown cells were adapted to these concentrations of ROS. Periodical addition of 100 μM H2O2 to pyrite cultures inoculated with pyrite-grown cells did not lower iron dissolution as it was observed with iron-grown cells. By high throughput proteomics analysis, an increased expression of proteins related to oxidative stress management, iron-and sulfur oxidation systems, carbon fixation and biofilm formation was observed in biofilm cells grown on pyrite compared to iron-grown cells.</jats:p

Notch Coordinates Periodontal Ligament Maturation through Regulating Lamin A

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Structure-Activity Relationships of Triple-Action Platinum(IV) Prodrugs with Albumin-Binding Properties and Immunomodulating Ligands

Chemotherapy with platinum complexes is essential for clinical anticancer therapy. However, due to side effects and drug resistance, further drug improvement is urgently needed. Herein, we report on triple-action platinum­(IV) prodrugs, which, in addition to tumor targeting via maleimide-mediated albumin binding, release the immunomodulatory ligand 1-methyl-d-tryptophan (1-MDT). Unexpectedly, structure–activity relationship analysis showed that the mode of 1-MDT conjugation distinctly impacts the reducibility and thus activation of the prodrugs. This in turn affected ligand release, pharmacokinetic properties, efficiency of immunomodulation, and the anticancer activity in vitro and in a mouse model in vivo. Moreover, we could demonstrate that the design of albumin-targeted multi-modal prodrugs using platinum­(IV) is a promising strategy to enhance the cellular uptake of bioactive ligands with low cell permeability (1-MDT) and to improve their selective delivery into the malignant tissue. This will allow tumor-specific anticancer therapy supported by a favorably tuned immune microenvironment

Weak Iron Oxidation by Sulfobacillus thermosulfidooxidans Maintains a Favorable Redox Potential for Chalcopyrite Bioleaching

Bioleaching is an emerging technology, describing the microbially assisted dissolution of sulfidic ores that provides a more environmentally friendly alternative to many traditional metal extraction methods, such as roasting or smelting. Industrial interest is steadily increasing and today, circa 15–20% of the world’s copper production can be traced back to this method. However, bioleaching of the world’s most abundant copper mineral chalcopyrite suffers from low dissolution rates, often attributed to passivating layers, which need to be overcome to use this technology to its full potential. To prevent these passivating layers from forming, leaching needs to occur at a low oxidation/reduction potential (ORP), but chemical redox control in bioleaching heaps is difficult and costly. As an alternative, selected weak iron-oxidizers could be employed that are incapable of scavenging exceedingly low concentrations of iron and therefore, raise the ORP just above the onset of bioleaching, but not high enough to allow for the occurrence of passivation. In this study, we report that microbial iron oxidation by Sulfobacillus thermosulfidooxidans meets these specifications. Chalcopyrite concentrate bioleaching experiments with S. thermosulfidooxidans as the sole iron oxidizer exhibited significantly lower redox potentials and higher release of copper compared to communities containing the strong iron oxidizer Leptospirillum ferriphilum. Transcriptomic response to single and co-culture of these two iron oxidizers was studied and revealed a greatly decreased number of mRNA transcripts ascribed to iron oxidation in S. thermosulfidooxidans when cultured in the presence of L. ferriphilum. This allowed for the identification of genes potentially responsible for S. thermosulfidooxidans’ weaker iron oxidation to be studied in the future, as well as underlined the need for new mechanisms to control the microbial population in bioleaching heaps

The Nrf1 CNC-bZIP Protein Is Regulated by the Proteasome and Activated by Hypoxia

BACKGROUND: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. PRINCIPAL FINDINGS: We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. CONCLUSIONS: Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status