No abstract

No abstract

Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4093-x) contains supplementary material, which is available to authorized users.

BackgroundEpidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear.MethodsTo investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs.ResultsExposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation.ConclusionsOur results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.

No abstract

Here we introduce a peptide model based on an alpha-helical coiled coil peptide, providing a simple system which can be used for a systematic study of the impact of different metal ions in different oxidation states on peptide secondary structure on a molecular level; histidine residues were incorporated into the heptad repeat to generate possible complexation sites for Cu2+ and Zn2+ ions.

No abstract

After reductions of fugitive and diffuse emissions by an industrial complex, a follow-up study was performed to determine the time variability of volatile organic compounds (VOCs) and the lifetime cancer risk (LCR). Passive samplers (3 M monitors) were placed outdoors (n = 179) and indoors (n = 75) in industrial, urban, and control areas for 4 weeks. Twenty-five compounds including n-alkanes, cycloalkanes, aromatics, chlorinated hydrocarbons, and terpenes were determined by GC/MS. The results show a significant decrease of all VOCs, especially in the industrial area and to a lesser extent in the urban area. The median outdoor concentration of benzene in the industrial area declined compared to the former study, around 85% and about 50% in the urban area, which in the past was strongly influenced by industrial emissions. Other carcinogenic compounds like styrene and tetrachloroethylene were reduced to approximately 60%. VOC concentrations in control areas remained nearly unchanged. According to the determined BTEX ratios and interspecies correlations, in contrast to the previous study, traffic was identified as the main emission source in the urban and control areas and showed an increased influence in the industrial area. The LCR, calculated for benzene, styrene, and tetrachloroethylene, shows a decrease of one order of magnitude in accordance to the decreased total VOC concentrations and is now acceptable according to values proposed by the World Health Organization.