We have successfully integrated MSe acquisitions into our proteomics experiments and have closely worked with bioinformatics group to validate our results on several projects. Mars-500 Project: This project is a collaboration with the State scientific center of the Russian Federation Institute for Bio-Medical Problems of RAS under the aegis of Roscosmos and Russian Academy of Sciences. This includes a series of experiments simulating long-term isolation of the crew of a Mars-bound space-flight. Plasma samples were from 6 healthy men (26-41 years old) and were obtained on the 14th and 7th day before, 17th, 51st and 85th day during and on the 7th day after a 105-day isolation in on-ground modules (Program MARS-500, http://mars500.imbp.ru/). Volunteers were selected after medical examination typical of cosmonauts. Because of the limited samples, standard rigorous statistical methods could not be applied, but a custom software algorithm written here at NIEHS by Shyamal Peddada and his group (Oriogen, Bioinformatics 2005 Oct 15;21(20)3933-4), detected a trend in some of the proteins quantified in this experiment suggesting a correlation between experimental conditions and the observed levels of these proteins. Effect of Bacillus anthracis lethal toxin (LT) on cardiomyocytes: Anthrax is an acute and lethal disease caused by the bacterium Bacillus anthracis which was used in biological attacks in 2001, and still potentially of use in bio-terrorism. It is still relatively unknown how exactly Anthrax toxin causes cell death, and there is no complete treatment for the disease. Anthrax toxin is composed of three major components: protective antigen (PA), edema factor (EF), and lethal factor (LF). When separated, each component is nontoxic. However, PA facilitates the entrance of EF and LF into cells, by combining with them to form dangerous toxins, edema toxin (EdTx) and lethal toxin (LeTx), respectively. While the effects of LeTx are documented in cell macrophages, additionally there have been reports of anthrax LeTx visibly damaging the heart tissue in mice. For example, a report from NIAID found that hearts from LeTx treated mice showed vacuole like spaces in the cardiac muscle tissue, with grainy punctuate staining the tissue. While the study was able to describe visual changes to the cardiomyocytes in the heart region of the mouse, there has been little investigation into the proteomic changes that may have taken place in these cells as well. Several families of proteins were observed to change in abundance upon LeTx treatment of cardiomyocytes. The most significant of these were three proteins involved in endocytosis and endosomal function. Two of the proteins are regulators of some forms of phosphatidyl-inositol phosphate, which are involved in a host of cell signaling processes, while FYVE domain proteins have been shown to mediate endosome activity and influence endocytosis, which is the chief method of LeTx entry into the cell. Also, a channel protein involved in apoptosis was seen to be up-regulated in cardiomyocytes. Several proteins involved in the regulation of cytochromes were down-regulated by the toxin, suggesting that mitochondria are compromised by toxin activity. Mitochondria have been shown to be sensitive to oxidative stress in cells. Toll like receptor 1 (TLR1), a TLR superfamily member, was also down-regulated by toxin action. The TLRs are involved in mediating the immune response. TLRs have also been shown to be affected by LeTx activity. Proteomic profiling of S-acylated macrophage proteins: S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have beeeen hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine vs. mock treatment conditions. Of 1,183 proteins identified in 4 independent experiments, 80 proteins were significant for S-acylation. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and 3 presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through 3H-palmitate labeling. Proteomics of paraffin-embedded tissues: As a continuation of a study of the effects of carbonyl sulfide inhalation exposure (in collaboration with R. Sills), we are investigating methodologies for proteomics studies of formalin-fixed paraffin-embedded (FFPE) tissues. Refinements of published protocols are needed to evaluate the suitability of the newly developed LC-MS-based label-free quantitative proteomics methodologies to the analysis of extracted FFPE tissue proteins from FFPE blocks. Initial experiments using FFPE brain tissues of six rats (6 males, 6 females) were carried out comparing extraction efficacies of proteins from FFPE tissues using home-made and published protocols. The best results were obtained when the tissue sections were deparaffinized in xylene and rehydrated through a series of alcohol treatments and when the protein extractions were carried out using an acid labile surfactant. Following these evaluations, triplicate samples of FFPE extracts will be analyzed and the proteins quantified. Overall, these preliminary results are encouraging and essentially indicate the applicability of LC-MS-based label-free quantitative proteomics methodologies to the analysis of extracted FFPE tissue proteins from FFPE blocks. Proteomics of LT-treated DMPO protected macrophages: Proteins from treated cells were separated by PAGE and in-gel digested for analysis by LC/MS/MS. Proteins were identified using Spectrum Mill. A Venn diagram was generated for proteins identified from each treated population. The number of proteins identified: Untreated, 468: LeTx, 327: DMPO, 341. Nitrotyrosine residues were observed for two proteins, DNAJb11 and Neurobeachin-like protein, from cells exposed to LeTx, but were not observed in the untreated or DMPO treated populations. These proteins may play a role in cell death through an oxidative stress mechanism.

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