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MALDI-TOF Analysis (maldi-tof + analysis)
Selected AbstractsDenitrifying bacteria anaerobically oxidize methane in the absence of ArchaeaENVIRONMENTAL MICROBIOLOGY, Issue 11 2008Katharina F. Ettwig Summary Recently, a microbial consortium was shown to couple the anaerobic oxidation of methane to denitrification, predominantly in the form of nitrite reduction to dinitrogen gas. This consortium was dominated by bacteria of an as yet uncharacterized division and archaea of the order Methanosarcinales. The present manuscript reports on the upscaling of the enrichment culture, and addresses the role of the archaea in methane oxidation. The key gene of methanotrophic and methanogenic archaea, mcrA, was sequenced. The associated cofactor F430 was shown to have a mass of 905 Da, the same as for methanogens and different from the heavier form (951 Da) found in methanotrophic archaea. After prolonged enrichment (> 1 year), no inhibition of anaerobic methane oxidation was observed in the presence of 20 mM bromoethane sulfonate, a specific inhibitor of MCR. Optimization of the cultivation conditions led to higher rates of methane oxidation and to the decline of the archaeal population, as shown by fluorescence in situ hybridization and quantitative MALDI-TOF analysis of F430. Mass balancing showed that methane oxidation was still coupled to nitrite reduction in the total absence of oxygen. Together, our results show that bacteria can couple the anaerobic oxidation of methane to denitrification without the involvement of Archaea. [source] Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galliFEBS JOURNAL, Issue 1 2005Rositsa Jordanova Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, ,-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N,-dimethyl- N -(iodoacetyl)- N,-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids. [source] The porcine trophoblastic interferon-,, secreted by a polarized epithelium, has specific structural and biochemical propertiesFEBS JOURNAL, Issue 11 2002Avrelija Cenci At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-,), together with lesser amounts of IFN-,, a unique species of type I IFN. This trophoblastic IFN-, (TrIFN-,) is an unprecedented example of IFN-, being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-, from other sources, either natural LeIFN-, (from adult leucocytes), or recombinant. Biologically active TrIFN-, is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-, which is formed of at least two polypeptide chains and four glycotypes. TrIFN-, collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N -acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l -fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-, molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-, on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-, a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium. [source] Synthesis and MALDI-TOF analysis of dendritic-linear block copolymers of lactides: Influence of architecture on stereocomplexationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 23 2006Alexandre Richez Abstract Formation of a stereocomplex from polylactide copolymers can be tuned by changing the size and the chain topology of the second block in the copolymer. In particular, the use of a dendritic instead of linear architecture is expected to destabilize the cocrystallisation of polylactide blocks. With this idea in mind, dendritic-linear block copolymers were synthesized by ring-opening polymerization (ROP) of lactides using benzyl alcohol dendrons of generation 1,3 as macroinitiators and stannous octoate as catalyst. Polymers with controlled and narrow molar mass distribution were obtained. The MALDI-TOF mass spectra of these dendritic-linear block copolymers show well-resolved signals. Remarkably, 10% or less of odd-membered polymers are present, indicating that ester-exchange reactions which occur classically parallel to the polymerization process, were in these conditions, very limited. Thermal analysis of polyenantiomers of generation 1,3 and the corresponding blends were examined. The blend of a pair of enantiomeric dendritic-linear block copolymers exhibit a higher melting temperature than each copolymer, characteristic for the formation of a stereocomplex. Melting temperatures are strongly dependent on the dendron generation. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 6782,6789, 2006 [source] Influence of Microwave Irradiation on the Lipase-Catalyzed Ring-Opening Polymerization of , -CaprolactoneMACROMOLECULAR RAPID COMMUNICATIONS, Issue 9 2006Patrick Kerep Abstract Summary: The microwave (MW)-assisted lipase-catalyzed ring-opening polymerization of , -caprolactone in boiling solvents was investigated for the first time. In case of boiling toluene or benzene the MW-assisted reaction proceeded significantly slower compared to oil bath heating. On the other hand, using boiling diethyl ether as solvent, an increase of the polymerization rate due to MW irradiation was found. Yield, molecular weight measurements, and MALDI-TOF analysis supported the results. Reactivity of the MW-assisted ring-opening polymerization of , -caprolactone compared with conventional thermal heating in different solvents. [source] Proteomic analysis of the response of an acidophilic strain of Chlamydomonas sp. (Chlorophyta) to natural metal-rich waterPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2010Cristina Cid Abstract A proteomic approach including 2-DE and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas sp. isolated from an extreme acidic environment, Río Tinto (southwest Spain). We have analyzed the soluble proteome obtained from whole cells growing on metal-rich natural acidic water from the river in comparison with the same strain growing in artificial BG-11 media. The most drastic effect was the decrease in the abundance of the ribulose-1,5-biphosphate carboxylase as well as other enzymes related to photosynthesis. However, phytochrome B, phosphoribulokinase, and phosphoglycerate kinase were upregulated when cells were grown in metal-rich acidic water. Besides, increased accumulation of two Hsps, Hsp70 and Hsp90 as well as other stress-related enzymes were also found in the cells growing in natural acidic water. These results suggest that naturally occurring metal-rich water induces a stress response in acidophilic Chlamydomonas forcing algal cells to reorganize their metabolic pathways as an adaptive response to these environmental conditions. [source] Differential Capture of Serum Proteins for Expression Profiling and Biomarker Discovery in Pre- and Posttreatment Head and Neck Cancer Samples,THE LARYNGOSCOPE, Issue 1 2008Gary L. Freed MD Abstract Introduction: A long-term goal of our group is to develop proteomic-based approaches to the detection and use of protein biomarkers for improvement in diagnosis, prognosis, and tailoring of treatment for head and neck squamous cell cancer (HNSCC). We have previously demonstrated that protein expression profiling of serum can identify multiple protein biomarker events that can serve as molecular fingerprints for the assessment of HNSCC disease state and prognosis. Methods: An automated Bruker Daltonics (Billerica, MA) ClinProt matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer was used. Magnetic chemical affinity beads were used to differentially capture serum proteins prior to MALDI-TOF analysis. The resulting spectra were analyzed using postprocessing software and a pattern recognition genetic algorithm (ClinProt 2.0). An HNSCC cohort of 48 sera samples from 24 patients consisting of matched pretreatment and 6 to 12 month posttreatment samples was used for further analysis. Low-mass differentially expressed peptides were identified using MALDI-TOF/TOF. Results: In the working mass range of 1,000 to 10,000 m/z, approximately 200 peaks were resolved for ionic bead capture approaches. For spectra generated from weak cation bead capture, a k-nearest neighbor genetic algorithm was able to correctly classify 94% normal from pretreatment HNSCC samples, 80% of pretreatment from posttreatment samples, and 87% of normal from posttreatment samples. These peptides were then analyzed by MALDI-TOF/TOF mass spectometry for sequence identification directly from serum processed with the same magnetic bead chemistry or alternatively after gel electrophoresis separation of the captured proteins. We were able to compare this with similar studies using surface-enhanced laser desorption ionization (SELDI)-TOF to show this method as a valid tool for this process with some improvement in the identification of our groups. Conclusions: This initial study using new high-resolution MALDI-TOF mass spectrometry coupled with bead fractionation is suitable for automated protein profiling and has the capability to simultaneously identify potential biomarker proteins for HNSCC. In addition, we were able to show improvement with the MALDI-TOF in identifying groups with HNSCC when compared with our prior data using SELDI-TOF. Using this MALDI-TOF technology as a discovery platform, we anticipate generating biomarker panels for use in more accurate prediction of prognosis and treatment efficacies for HNSCC. [source] Age of SERPINA1 Gene PI Z Mutation: Swedish and Latvian Population AnalysisANNALS OF HUMAN GENETICS, Issue 3 2008B. Lace Summary Alpha 1-antitrypsin (A1AT) deficiency, one of the most common inborn errors of metabolism in Caucasians, is characterized by a low serum concentration of A1AT and a high risk of pulmonary emphysema and liver disease. The allelic frequency for the most common protease inhibitor (PI) Z mutation in the SERPINA1 gene is 2,5% in Caucasians of European descent. The objective of our study was to estimate the PI Z mutation age using molecular analysis in Latvian and Swedish populations, which have the highest frequency of PI Z mutation. DNA samples of heterozygous and homozygous PI Z allele carriers from Latvia (n = 21) and Sweden (n = 65) were analysed; 113 unrelated healthy donors from Latvia were used as a control group. MALDI-TOF analysis was performed on all samples. Pairwise Fst was computed to compare the PI Z mutation ages between the two populations and controls. A p value less than 0.05 was considered significant. Analysis of non-recombinant SNPs revealed that the PI Z mutation age was 2902 years in Latvia (SD 1983) and 2362 years in Sweden (SD 1614) which correlates with previous studies based on microsatellite analysis. [source] New Peptolides from the Cyanobacterium Nostoc insulare as Selective and Potent Inhibitors of Human Leukocyte ElastaseCHEMBIOCHEM, Issue 16 2008Christian Mehner Abstract Eight new cyanopeptolins (insulapeptolides A,H) were obtained from the cyanobacterium Nostoc insulare. Their isolation was guided by their bioactivity toward the target enzyme human leukocyte elastase, molecular biological investigations, and MALDI-TOF analysis. These peptides are selective inhibitors of human leukocyte elastase with activities in the nanomolar range. Insulapeptolide D (4) was the most potent compound with an IC50 value of 85 nM (Ki value of 36 nM). [source] Star polymers by cross-linking of linear poly(benzyl- L -glutamate) macromonomers via free-radical and RAFT polymerization.JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 20 2010A simple route toward peptide-stabilized nanoparticles Abstract Poly(benzyl- L -glutamate) (PBLG) macromonomers were synthesized by N -carboxyanhydride (NCA) polymerization initiated with 4-vinyl benzylamine. MALDI-ToF analysis confirmed the presence of styrenic end-groups in the PBLG. Free-radical and RAFT polymerization of the macromonomer in the presence of divinyl benzene produced star polymers of various molecular weights, polydispersity, and yield depending on the reaction conditions applied. The highest molecular weight (Mw) of 10,170,000 g/mol was obtained in a free-radical multibatch approach. It was shown that the PBLG star polymers can be deprotected to obtain poly(glutamic acid) star polymers, which form water soluble pH responsive nanoparticles. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010 [source] | ||||||||||||||||