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MALDI-TOF Mass Spectrometry (maldi-tof + mass_spectrometry)
Selected AbstractsSample Target Substrates with Reduced Spot Size for MALDI-TOF Mass Spectrometry Based on Patterned Self-Assembled MonolayersADVANCED FUNCTIONAL MATERIALS, Issue 17 2009Nicole Herzer Abstract The wetting properties of structured self-assembled monolayers are used to fabricate sample target substrates for MALDI-TOF mass spectrometry. Combining the advantages of a hydrophobic-hydrophilic surface pattern and the possibility of obtaining micrometer patterns allows an increase in the sensitivity of MALDI-TOF mass spectrometry analysis and a reduction in the traceable concentration down to fmol µL,1. This easy, cheap and fast patterning process provides substrates that allow sensitive, high-resolution mass spectrometry of dilute solutions. [source] Structural Studies of the O-Chain Polysaccharide from Plesiomonas shigelloides Strain 302,73 (Serotype O1)EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 18 2008Giuseppina Pieretti Abstract Plesiomonas shigelloides is a Gram-negative bacterium belonging to the Enterobacteriaceae family. It has been found in an aquatic environment in the tropical and subtropical regions and is responsible for many gastrointestinal infections in humans, which take place from drinking untreated water or eating uncooked shellfish. Plesiomonas shigelloides has also been reported to provoke extraintestinal infections such as meningitis and bacteremia in immunocompromised adults and neonates. Despite the emerging importance of this pathogenic microorganism, only three different O-antigens have been characterised so far. The structure of the O-chain of the lipopolysaccharide (LPS) from Plesiomonasshigelloides strain 302,73 (serotype O1) was determined by chemical analysis, 1D and 2D NMR spectroscopy and MALDI-TOF mass spectrometry. The polysaccharide was constituted by a linear pentasaccharidic repeating unit as follows: ,3)-,- L -PneNAc4OAc(1,4)-,- L -FucNAc(1,4)-,- L -FucNAc(1,4)-,- L -FucNAc(1,3)-,- D -QuiNAc4NHb(1, (PneNAc = 2-acetamido-2,6-dideoxy-talose, Hb = (S)-3-hydroxybutanoyl) PneNAc O -acetylation was not stoichiometric and was found to be about 75,%. The position of the O -acetyl group and the amount of acetylation were deduced by NMR spectroscopic analysis. All the monosaccharides included in the repeating unit were deoxyamino sugars, which most probably, together with the presence of O -acetyl groups, were responsible for the recovery of the LPS in the phenol layer of the phenol/water extract of dried bacteria cells.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] Systematic Studies on Photoluminescence of Oligo(arylene-ethynylene)s: Tunability of Excited States and Derivatization as Luminescent Labeling Probes for ProteinsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2006Yong-Gang Zhi Abstract Functionalized oligo(phenylene-ethynylene)s (OPEs) with different conjugation lengths, p -X(C6H4C,C)nSiMe3 (n = 1,4; X = NH2, NMe2, H) were synthesized by Sonogashira coupling of (phenylene-ethynylene)s and 1-iodo-4-(trimethylsilylethynyl)benzene, followed by desilylation of the p -substituted (trimethylsilylethynyl)benzenes with potassium hydroxide. The photoluminescent properties for the OPE series with different chain lengths and their solvatochromic responses were examined. The absorption maxima were red-shifted with increasing numbers of ,(C6H4C,C), units (n), and a linear plot of the absorption energy maxima vs. 1/n was obtained for each series. The emission spectra in dichloromethane showed a broad and structureless band, the energies of which (in wavenumbers) also fit linearly with 1/n. Both the absorption and emission wavelength maxima of the NH2 - and NMe2 -substituted OPEs exhibited significant solvent dependence, whereas the parent OPEs (X = H) showed only minor shifts of the ,max values in different solvents. Substituent effects upon the photoluminescent characteristics of the OPEs and the tunability of the excited states were examined with the p -X(C6H4C,C)nSiMe3 (n = 2, 3; X = NH2, NMe2, H, SMe, OMe, OH, and F) series. The H- and F-substituted counterparts exhibited high-energy vibronically structured emissions attributed to the 3(,,*) excited states of the (arylene-ethynylene) backbone. For compounds bearing NH2 and NMe2 groups, a broad red-shifted emission with a remarkable Stokes shift from the respective absorption maximum was observed, which can be assigned to an n , ,* transition. The n , ,* assignment was supported by MO calculations on the model compounds p -X(C6H4C,C)2SiH3 (X = NH2, H). Functionalization of the oligo(arylene-ethynylene)s with the N -hydroxysuccinimidyl (NHS) moiety enabled covalent attachment of the fluorophore to HSA protein molecules. A series of fluorescent labels, namely p -X(C6H4C,C)nC6H4NHS, (n = 1, X = NH2, NMe2, SMe, OMe, OH, F; n = 2, X = NH2, NMe2) and p -Me2NC6H4C,C(C4H2S)C,CC6H4NHS were synthesized, and their conjugates with HSA (human serum albumin) were characterized by MALDI-TOF mass spectrometry, UV/Vis absorption spectroscopy, and gel electrophoresis. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] The use of ITS DNA sequence analysis and MALDI-TOF mass spectrometry in diagnosing an infection with Fusarium proliferatumEXPERIMENTAL DERMATOLOGY, Issue 11 2008Florian Seyfarth Abstract:, Although mycoses are among the most common diseases worldwide, infections with Fusarium spp. occur only rarely. Mostly patients suffering from underlying immune deficiency are infected with this mould, resulting in a considerably decreasing prognosis. In immunocompromised patients, cutaneous manifestations are more often associated with Fusarium sp. than with Candida sp. or Aspergillus sp. We describe one patient with acute lymphoblastic leukaemia, who was first treated with chemotherapy after GMALL protocol 07/03. After relapse, the patient was successfully transplanted in second remission with a human leukocyte antigen (HLA)-matched unrelated peripheral blood stem cell graft. Ten months later, the patient died from respiratory insufficiency and recurrence of leukaemia. Previously, Aspergillus antigen was detected in blood. In the latter course, disseminated papules appeared. One of these was examined histologically and mycologically. Conventional cultural diagnostics led to the diagnosis of a fusariosis, further supported by internal transcribed spacer (ITS) sequencing and matrix assisted laser desorption/ionisation,time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry, both determining the isolated strain as Fusarium proliferatum, which is a very infrequent pathogen within this genus. Our investigations underline the potential of MALDI-TOF MS based identification of Fusarium species as an innovative, time and cost efficient alternative to ITS sequencing. [source] Complete subunit sequences, structure and evolution of the 6 × 6-mer hemocyanin from the common house centipede, Scutigera coleoptrataFEBS JOURNAL, Issue 13 2003Kristina Kusche Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure,function relationship and evolution. S. coleoptrata hemocyanin is a 6 × 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5,55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 × 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata. [source] Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylaseFEBS JOURNAL, Issue 5 2003Structural, functional implications Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302,6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19,33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15,K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein- l -isoaspartic acid O -methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR,,34 min to ,,31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR,,34 min species decreased with a biphasic time-course with t0.5 -values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ,,1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial ,-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32,Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition. [source] Sample Target Substrates with Reduced Spot Size for MALDI-TOF Mass Spectrometry Based on Patterned Self-Assembled MonolayersADVANCED FUNCTIONAL MATERIALS, Issue 17 2009Nicole Herzer Abstract The wetting properties of structured self-assembled monolayers are used to fabricate sample target substrates for MALDI-TOF mass spectrometry. Combining the advantages of a hydrophobic-hydrophilic surface pattern and the possibility of obtaining micrometer patterns allows an increase in the sensitivity of MALDI-TOF mass spectrometry analysis and a reduction in the traceable concentration down to fmol µL,1. This easy, cheap and fast patterning process provides substrates that allow sensitive, high-resolution mass spectrometry of dilute solutions. [source] Amphiphilic Homochiral Oligopeptides Generated via Phase Separation of Nonracemic , -Amino Acid Derivatives and Lattice-Controlled Polycondensation in a Phospholipid EnvironmentHELVETICA CHIMICA ACTA, Issue 11 2003Irina Rubinstein Racemic S -ethyl thioesters of N, -stearoyllysine (=,S -ethyl (R,S)-2-amino-6-(stearoylamino)hexanethioate) and S -ethyl thioesters of , -stearyl glutamic acid (=stearyl (R,S)-4-amino-5-(ethylsulfanyl)-5-oxopentanoate) self-assemble as separated two-dimensional crystalline monolayers within an achiral phospholipid environment of racemic 1,2-dipalmitoylglycerol (DPG) and 1,2-dipalmitoylglycero-3-phosphoethanolamine (DPPE), as demonstrated by grazing-incidence X-ray-diffraction (GIXD) measurements performed on the surface of H2O. Lattice-controlled polycondensation within these crystallites with deuterium-enantiolabeled monomers was initiated by injecting aqueous solutions of Ag+ or I2/KI beneath the monolayers, which yielded mixtures of diastereoisomeric oligopeptides containing up to six to eight repeating units, as analyzed by MALDI-TOF mass spectrometry. Analysis of the diastereoisomeric distribution showed an enhanced relative abundance of the oligopeptides with homochiral sequences containing three or more repeating units. Within the DPPE monolayers, the nucleophilic amino group of the phospholipid operates as an initiator of polymerization at the periphery of the monomer two-dimensional crystallites. Enhanced relative abundance of enantiomerically enriched homochiral oligopeptides was obtained by the polycondensation of nonracemic monomers. This enhancement indicated a phase separation into racemic and enantiomorphous monomer crystallites within the phospholipid environment, although this separation could not be observed directly by GIXD. A possible role that might have been played by crystalline assemblies for the abiotic generation and amplification of oligopeptides with homochiral sequences is discussed. [source] Novel PlexorÔ SNP genotyping technology: comparisons with TaqMan® and homogenous MassEXTENDÔ MALDI-TOF mass spectrometry,HUMAN MUTATION, Issue 9 2007E.A. Tindall Abstract Analysis of SNPs for association, linkage, haplotype, and pharmacogenetic studies has led to a dramatic increase in the number and evolution of medium- to high-throughput genotyping technologies. This study introduces PlexorÔ as a new method for medium-throughput (single SNP) genotyping. We compare this fluorescent-based chemistry for call rate, accuracy, affordability, throughput, and overall efficiency against two commonly used technologies. These include fluorescent-based TaqMan® allelic discrimination for single SNP analysis (medium-throughput) and the homogenous MassEXTENDÔ (hMEÔ) chemistry using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for multiple SNP analysis (high-throughput). Analysis of 11 SNPs, including all six possible nucleotide substitutions, showed PlexorÔ to be highly comparable for both call rate (94.7%) and accuracy (99.2%) to the TaqMan® (94.6% and 99.8%, respectively) and hMEÔ (91.9% and 98.1%, respectively) chemistries. We demonstrate that this novel method is an efficient, cost-effective alternative to TaqMan® genotyping commonly used in diagnostic settings. Hum Mutat 28(9), 922,927, 2007. © 2007 Wiley-Liss, Inc. [source] Overexpression of Lysyl Hydroxylase-2b Leads to Defective Collagen Fibrillogenesis and Matrix Mineralization,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2005Suchaya Pornprasertsuk Abstract Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. Introduction: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. Materials and Methods: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. Results: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. Conclusions: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone. [source] Isolation and Identification of Low-Molecular-Weight Peptides from Emmentaler CheeseJOURNAL OF FOOD SCIENCE, Issue 2 2002C. Combes ABSTRACT: Four peptides, derived from the N-terminal fragment of ,sl -casein [CN (1,9), CN (1,12), CN (1,13) and CN (1,14)] were isolated from a low-molecular-weight extract of a commercial Emmentaler cheese (age: approx. 5.5 mo) by gel filtration and RP-FPLC. Capillary zone electrophoresis was used to check the purity of the peptides. Using automatic Edman degradation and MALDI-TOF mass spectrometry, amino acid sequence and molecular mass of the peptides were determined to identify them. The peptide ,sl -CN (1,13) was identified for the first time in Emmentaler cheese. The peptide fraction was shown not to contribute to the characteristic flavor of Emmentaler cheese. [source] The identification of synthetic homopolymer end groups and verification of their transformations using MALDI-TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2010Yejia Li Abstract Recent advances in the resolving power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) enable the detailed characterization of linear homopolymers, and in particular provide invaluable data for the determination of their end-group functionalities. With the growing importance of macromolecular coupling reactions in building complex polymer architectures, the ability to accurately monitor end-group transformations is becoming increasingly important for synthetic polymer chemists. This tutorial demonstrates the application of MALDI-TOF MS in determining both end-group functionalities and their transformations for linear homopolymers. Examples of both polycaprolactone and polystyrene are examined, and the strengths and weaknesses of various approaches to data analysis are given. Copyright © 2010 John Wiley & Sons, Ltd. [source] MALDI-TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varietiesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2009alplachta Abstract A procedure for identification of malting barley varieties using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ethanol-soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI-TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry-based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd. [source] Characterization of natural wax esters by MALDI-TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2009Vladimír Vrkoslav Abstract The applicability of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to the analysis of wax esters (WEs) was investigated. A series of metal salts of 2,5-dihydroxybenzoic acid (DHB) was synthesized and tested as possible matrices. Alkali metal (Li, Na, K, Rb, Cs) and transition metal (Cu, Ag) salts were studied. The matrix properties were evaluated, including solubility in organic solvents, threshold laser power that should be applied for successful desorption/ionization of WEs, the nature of the matrix ions and the mass range occupied by them, and the complexity of the isotope clusters for individual metals. Lithium salt of dihydroxybenzoic acid (LiDHB) performed the best and matrices with purified lithium isotopes (6LiDHB or 7LiDHB) were recommended for WEs. Three sample preparation procedures were compared: (1) mixing the sample and matrix in a glass vial and deposition of the mixture on a MALDI plate (Mix), (2) deposition of sample followed by deposition of matrix (Sa/Ma), and (3) deposition of matrix followed by deposition of sample (Ma/Sa). Morphology of the samples was studied by scanning electron microscopy. The best sample preparation technique was Ma/Sa with the optimum sample to matrix molar ratio 1 : 100. Detection limit was in the low picomolar range. The relative response of WEs decreased with their molecular weight, and minor differences between signals of saturated and monounsaturated WEs were observed. MALDI spectra of WEs showed molecular adducts with lithium [M + Li]+. Fragments observed in postsource decay (PSD) spectra were related to the acidic part of WEs [RCOOH + Li]+ and they were used for structure assignment. MALDI with LiDHB was used for several samples of natural origin, including insect and plant WEs. A good agreement with GC/MS data was achieved. Moreover, MALDI allowed higher WEs to be analyzed, up to 64 carbon atoms in Ginkgo biloba leaves extract. Copyright © 2008 John Wiley & Sons, Ltd. [source] MALDI-TOF mass spectrometric analysis for the characterization of the 5,10,15,20-tetrakis- (m -hydroxyphenyl)bacteriochlorin (m -THPBC) photoproducts in biological environmentJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005Henri-Pierre Lassalle Abstract Photoproducts formation upon irradiation (739 nm) of 5,10,15,20-tetrakis(m -hydroxyphenyl)bacteriochlorin (m -THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption spectroscopy and MALDI-TOF mass spectrometry. The experiments were performed with a freshly prepared PBS,HSA solution of m -THPBC and with a PBS,HSA m -THPBC solution incubated for 6 h at 37 °C. The incubation of m -THPBC solution leads to the dye monomerisation, whereas in the freshly prepared solution, m -THPBC is under an aggregated form. Regardless of the incubation condition, photobleaching experiments carried out by absorption spectroscopy demonstrate the degradation of the photosensitizer and its phototransformation in m -THPC. Moreover, m -THPC was the sole photoproduct detected using absorption spectroscopy. Together with a degradation of m -THPBC and formation of m -THPC, MALDI-TOF mass spectrometry evidenced several other photoinduced modifications. Photoproducts such as dihydroxy m -THPBC and dihydroxy m -THPC were detected in both conditions; however, the formation of hydroxylated photoproducts was significantly greater in incubated solution. In addition, small molecules arising from the degradation of the photosensitizer and identified as dipyrin derivatives and dipyrrolic synthon were observed. Copyright © 2005 John Wiley & Sons, Ltd. [source] Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2005Akihito Ishigami Abstract Citrullinated proteins are the products of a posttranslational process in which arginine residues undergo modification into citrulline residues when catalyzed by peptidylarginine deiminases (PADs) in a calcium ion-dependent manner. In our previous report, PAD2 expressed mainly in the rat cerebrum became activated early in the neurodegenerative process. To elucidate the involvement of protein citrullination in human neuronal degeneration, we examined whether citrullinated proteins are produced during Alzheimer's disease (AD). By Western blot analysis with antimodified citrulline antibody, citrullinated proteins of varied molecular weights were detected in hippocampal tissues from patients with AD but not normal humans. Two of the citrullinated proteins were identified as vimentin and glial fibrillary acidic protein (GFAP) by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Interestingly, PAD2 was detected in hippocampal extracts from AD and normal brains, but the amount of PAD2 in the AD tissue was markedly greater. Histochemical analysis revealed citrullinated proteins throughout the hippocampus, especially in the dentate gyrus and stratum radiatum of CA1 and CA2 areas. However, no citrullinated proteins were detected in the normal hippocampus. PAD2 immunoreactivity was also ubiquitous throughout both the AD and the normal hippocampal areas. PAD2 enrichment coincided well with citrullinated protein positivity. Double immunofluorescence staining revealed that citrullinated protein- and PAD2-positive cells also coincided with GFAP-positive cells, but not all GFAP-positive cells were positive for PAD2. As with GFAP, which is an astrocyte-specific marker protein, PAD2 is distributed mainly in astrocytes. These collective results, the abnormal accumulation of citrullinated proteins and abnormal activation of PAD2 in hippocampi of patients with AD, strongly suggest that PAD has an important role in the onset and progression of AD and that citrullinated proteins may become a useful marker for human neurodegenerative diseases. © 2005 Wiley-Liss, Inc. [source] Resin comparison and fast automated stepwise conventional synthesis of human SDF-1,JOURNAL OF PEPTIDE SCIENCE, Issue 12 2008Hirendra Patel Abstract Human SDF-1, contains 68 amino acids and is a member of the chemokine family of peptides. This long peptide was synthesized stepwise using classical conditions in 101 h. The reaction times were then reduced to deprotection times of 2 × 2 min and coupling times of 2 × 2.5 min, resulting in a total synthesis time of 22 h. The effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities was compared. A small portion of crude peptide was purified using an RP-HPLC column and the mass of the final product was confirmed with MALDI-TOF mass spectrometry. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Facile detection of specific RNA-polypeptide interactions by MALDI-TOF mass spectrometryJOURNAL OF PEPTIDE SCIENCE, Issue 8 2008Maki Sugaya Abstract A simple method for the detection of specific RNA-polypeptide interactions using MALDI-TOF mass spectroscopy is described. Instead of direct observation of the RNA-polypeptide complex, we attempted the indirect observation of the binding event by focusing on the disappearance of the free polypeptide signal upon interaction with RNA. As a result, specific binding of the Rev-response element (RRE) RNA of the HIV with two RRE-binding peptide aptamers, DLA and RLA peptides, as well as the bacteriophage , boxB RNA with the , N peptide was observed. We also show that specific RNA-binding peptides can be identified from a mixture of peptides with varying RNA-binding affinity, showing that the method could be applied to high-throughput screening from simple peptide libraries. The method described in this study provides a quick and simple method for detecting specific RNA,polypeptide interactions that avoids difficulties associated with direct observation of RNA and RNA,polypeptide complexes, which may find various applications in the analysis of RNA,polypeptide interactions and in the identification of novel RNA-binding polypeptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Location of Disulfide bonds in mature ,- L;-fucosidase from peaJOURNAL OF PEPTIDE SCIENCE, Issue 6 2001Anna Codina Abstract Fuc-9 is the mature form of a vacuolar ,- L;-fucosidase enzyme which seems to play an important role in plant growth regulation. Fuc-9 is a 202-residue protein containing five Cys residues located at positions 64, 109, 127, 162 and 169. In this study, the disulfide structure of Fuc-9 was determined by MALDI-TOF mass spectrometry (MS), with minimal clean-up of the samples and at a nanomolar scale. Two strategies, based on a specific chemical cleavage (with 2-nitro-5-thiocyanobenzoic acid and alkaline conditions) at the Cys residues and modification of Cys residues by acrylamide/deuterium labeled acrylamide alkylation, were used. Using these methods, the disulfide pairings Cys64-Cys109 and Cys162-Cys169 could be established. The advantages and limitations of our experimental approach are discussed. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Synthesis of phenanthroline-terminated polymers and their Fe(II)-complexesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 12 2010Miklós Nagy Abstract Well-defined mono- and bifunctional, phenanthroline-terminated poly(ethylene glycol) and polyisobutylene capable of polymer network formation were synthesized. The starting materials mono- and bi-phenanthroline- (phen) terminated poly(ethylene glycols) (mPEG-phen, phen-PEG-phen) and polyisobutylenes (PIB-phen, phen-PIB-phen) were prepared by the Williamson synthesis and characterized by means of 1H NMR and MALDI-TOF mass spectrometry. According to UV,Vis spectrophotometry and ESI-TOF mass spectrometry, the phenanthroline-terminated polymers underwent quantitative complex formation with ferrous ions in solution. The aqueous solution of mPEG-phen shows self-assembly behavior. Important parameters, such as critical micelle concentration and hydrodynamic radius of the aggregates were also determined. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2709,2715, 2010 [source] Syntheses of multicyclic poly(ether sulfone)s from 5,5,,6,6,-tetrahydroxy-3,3,3,,3,-tetramethyl spirobisindane and 4,4,-bis(4-chlorophenyl) sulfonesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 11 2008Hans R. Kricheldorf Abstract 5,5,,6,6,-Tetrahydroxy-3,3,3,,3,-tetramethyl spirobisindane (TTSBI) was polycondensed with 4,4,-dichlorodiphenyl sulfone (DCDPS) or with 4,4,-bis(4-chlorophenyl sulfonyl) biphenyl (BCSBP) in DMSO. Concentration and feed ratio were optimized to avoid gelation and to obtain a maximum yield of multicyclic polyethers free of functional groups. Regardless of these reaction conditions, only low fractions of perfect multicycles were obtained from DCDPS apparently due to steric hindrance of ring closure. Under the same conditions high fractions of perfect multicycles were achieved with the longer and more flexible DCSBP. The reaction products were characterized by MALDI-TOF mass spectrometry, 1H-NMR spectroscopy viscosity, and DSC measurements. Relatively low glass transition temperatures (Tgs , 160,175 °C) were found. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3732,3739, 2008 [source] Polyimides based on new diamines having pendant imide groupsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 24 2005Hans R. Kricheldorf Abstract New aromatic diamines were prepared in two steps from 4,5-dichlorophthalic anhydride and primary amines. The resulting 4,5-dichlorophthalimide was reacted with 4-mercaptoaniline, so that the chloroatoms were substituted by the mercapto groups (via the sulfide anions). The new diamines were polycondensed either with the diphenyl ether 3,3,,4,4,-tetracarboxylic anhydride or with bicyclooctane tetracarboxylic anhydride. These polycondensations were conducted in boiling m -cresol with azeotropic removal of water. The isolated polyimides were characterized by viscosity measurement, IR-spectroscopy, elemental analyses, and MALDI-TOF mass spectrometry. The mass spectra evidenced a high content of cyclic polyimides, indicating nearly perfect reaction conditions. The mass spectra also proved the formation of copolymers containing one diamine with a trialkylamine group in the side chain. High glass transition temperatures but a low crystallization tendency were found by DSC measurements. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 6272,6281, 2005 [source] Syntheses of trimethoxysilyl-endcapped polylactones via 3-mercaptopropyl trimethoxysilane,JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 16 2005Hans R. Kricheldorf Abstract Monofunctional polylactones were prepared by Bu2Sn(OMe)2 -initiated ring-opening polymerization of ,-caprolactone (,CL) followed by acylation with bromoacetylbromide. Telechelic polylactones and polylactides were prepared via ring-expansion polymerization with 2,2-dibutyl-2-stanna-1,3-dioxepane (DSDOP) or 2,2-dibutyl-2-stanna-pentaoxacyclotridecane (Bu2SnTEG) as cyclic initiator. In situ combination of the polymerization with condensation by means of bromoacetylbromide yielded polylactones having bromoacetate endgroups. These endgroups were subjected to nucleophilic substitution with 3-mercaptopropyl trimethoxysilane (3-MPTMS). Analogous experiments were conducted with dl-lactide. The telechelic trimethoxysilyl-endcapped polylactones were characterized by viscosity, 1H and 13C NMR-spectroscopy, and MALDI-TOF mass spectrometry. The mass spectra revealed small amounts of cyclic oligolactones as byproducts in all samples. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3667,3674, 2005 [source] Multicyclic Poly(ether sulfone)s Derived from Tris(4-hydroxyphenyl)ethaneMACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 7 2007Mazen Garaleh Abstract Polycondensations of 4,4,-difluorodiphenylsulfone with tris(4-hydroxy phenyl)ethane were performed in DMSO with variation of feed ratio and concentration. For feed ratios of 1.0:1.3,1.0:1.5, soluble multicyclic poly(ether sulfone)s were obtained when the monomer concentration was below 0.05 M. The conversions were never complete under standard conditions, and doubling the reaction time yielded perfect multicyclic products free of endgroups; however, a small fraction of the product was crosslinked under these conditions. Quite similar results were obtained with 4,4,-dichlorodiphenylsulfone at higher temperatures. When K2CO3 was replaced by tertiary amines, conversions and molecular weights were lower. The multicyclic poly(ether sulfone)s were characterized by MALDI-TOF mass spectrometry, SEC, and DSC measurements. Broad molecular weight distributions with polydispersities in the range of 2.4,3.9 were found, and, surprisingly, two glass transitions were detectable in the DSC heating curves. [source] New Polymer Syntheses, 112MACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 4 2003Hans R. Kricheldorf Abstract Bis(4-chlorophenyl)sebacate (BCPS) was polycondensed with 1,12-diamino-4,9-dioxadodecane or with its bistrimethylsilyl derivative in three different solvents with variation of time and temperature. The highest molecular weights were obtained in dimethylsulfoxide at 60,°C. The highest fraction of cyclic polyamides was detected by MALDI-TOF mass spectrometry in the samples with the highest molecular weights. Numerous polycondensations of BCPS and 1,13-diamino-4,7,10-trioxatridecane were performed with variation of solvent, time and temperature. Again the best results were obtained in dimethylsulfoxide at 60,°C. The fraction of cyclic polyamides increased with higher average molecular weights of the samples. The MALDI-TOF mass spectrum of the sample with the highest molar mass (Mn ca. 30,000 Da) exclusively displayed mass peaks of cycles (detected up to 13,000 Da). No side reactions were observed. MALDI-TOF mass spectrum (segment) of polyamide 1 prepared by polycondensation of DDD with BCPS in DMSO at 60,°C/48 h (no. 3, Table 1). [source] Supramolecular Nanocycles Comprising , -Cyclodextrin-click-Ferrocene Units: Rings of Rings of RingsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 7 2010Maricica Munteanu Abstract We applied 1,3-dipolar cycloaddition to bind ethynylferrocene onto 6I-azido-6I-deoxycyclomaltoheptaose under microwave assisted conditions. The process was investigated by 1H NMR, FT-IR spectroscopy, and MALDI-TOF mass spectrometry. The ability of the synthesized compound to self-organize to cyclic supramolecular structures was investigated by dynamic light scattering measurements and cryo-transmission electron microscopy. [source] Uncharged Water-Soluble Metal-Bis-Porphyrins Like Molecular Tweezers for Amino AcidsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 15 2007Emilio Scamporrino Abstract Some new water-soluble bis-porphyrins, constituted of two porphyrin units spaced by means of aliphatic bridges of different lengths, were synthesized and characterized by MALDI-TOF mass spectrometry, 1H NMR and UV-vis spectroscopy. The hydrosolubility of these uncharged compounds was guaranteed from the presence of six long PEG chains bound on the peripheral positions of the two porphyrins. Cobalt and zinc derivatives were also prepared. In the case of Co-bis-porphyrin, the appearance of induced circular dichroism (ICD) signals in water solution confirmed the formation of stable complexes with some amino acids, in which the bis-porphyrin behaves like molecular tweezers. [source] Additional Retardation in RAFT Polymerization: Detection of Terminated Intermediate RadicalsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 7 2007Maël Bathfield Abstract The reversible addition-fragmentation chain transfer (RAFT) polymerization of N -acryloylmorpholine (NAM) is performed using three dithioesters (DT) as chain transfer agents (CTA) that incorporate a morpholine (morpholine-DT), a biotin (biotin-DT), or a sugar (sugar-DT) moiety in the R group. PolyNAM chains of controlled characteristics are synthesized. An unexpected behavior is observed with morpholine-DT, described as an ,additional retardation', which is especially visible when low molar masses are targeted (,<,5,000 g,·,mol,1). In that particular case, further investigations using MALDI-TOF mass spectrometry show the presence of terminated intermediate radicals (IRs), which corroborates the assumption based on a specific protection of IR according to the nature of the , -chain-end. [source] Cyclic Polymers by Kinetically Controlled Step-Growth PolymerizationMACROMOLECULAR RAPID COMMUNICATIONS, Issue 5-6 2003Hans R. Kricheldorf Abstract The theory of step-growth polymerizations including the cascade theory is discussed in the light of new results focussing on the role of cyclization reactions. The identification of cyclic oligomers and polymers in reaction products of step-growth polymerizations has been eased considerably by means of MALDI-TOF mass spectrometry. Experimental examples concern syntheses of polyesters, polycarbonates, polyamides, polyimides, poly(ether sulfone)s, poly(ether ketone)s and polyurethanes. It was found in all cases that the percentage and molecular weight of the cycles increases when the reaction conditions favor high molecular weights. In the absence of side reactions all reaction products will be cycles when conversion approaches 100%. Cyclization may even take place in the nematic phase but even-numbered cycles are favored over odd-numbered ones due to electronic interactions between mesogens aligned in parallel. In contrast to Flory's cascade theory, cyclization also plays a decisive role in polycondensations of abn -type monomers, and at 100% conversion all hyperbranched polymers have a cyclic core. Furthermore, it is demonstrated that in a2+b3 polycondensations intensive cyclization in the early stages of the process has the consequence that either no gelation occurs or the resulting networks consist of cyclic and bicyclic oligomers as building blocks. Finally, a comparison between cyclization of synthetic polymers and biopolymers is discussed. Schematic representation of a network structure mainly consisting of cyclic oligomers and multicyclic building blocks as derived from "a2" + "b3" polycondensation. [source] Protein fraction isolated from epididymal fluid re-associates sperm in vitro: Possible role of serpins in rat rosettes assemblyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2010María A. Monclus Abstract In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an ,-1 antitrypsin and a new protein with an ,-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm,proteases interaction was proposed. Mol. Reprod. Dev. 77: 410,419, 2010. © 2010 Wiley-Liss, Inc. [source] | |||||||||||||||||||