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MALDI-TOF
Terms modified by MALDI-TOF Selected AbstractsEffect of celecoxib on cyclooxygenase-2 expression and possible variants in a patient with Barrett's esophagusDISEASES OF THE ESOPHAGUS, Issue 3 2007G. A. Jacobson SUMMARY., Cyclooxygenase-2 (COX-2) expression is increased in metaplastic and dysplastic Barrett's esophageal epithelium and it is thought that selective COX-2 inhibitors could offer hope as chemoprevention therapy. The aim of the study was to investigate the in vivo effect of celecoxib on COX-2 expression in patients with Barrett's esophagus and no recent history of non-steroidal anti-inflammatory drug use. Endoscopic mucosal biopsy specimens were collected at baseline and after 28 days of therapy in a patient treated with celecoxib 200 mg twice daily. Samples were analyzed for COX-2 expression by immunoblot analysis with chemiluminescence detection. COX-2 expression was found to decline 20% and 44% at two different biopsy sites compared to the baseline sample. Longer exposures revealed a number of previously unidentified proteins above and below the 67 kDa COX-2 protein including 38 kDa and 45 kDa proteins which were present only at study completion consistent with up-regulation after celecoxib therapy. Further investigations of the 38 kDa and 45 kDa proteins were undertaken using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with immunoblot and MALDI-TOF (matrix assisted laser desorption ionization , time of flight) analysis but no matches were found and results were inconclusive. Unmatched masses from MALDI-TOF peptide mass fingerprinting were compared with human COX-2 (67 kDa) and COX-2b (39 kDa) using unspecific cleavage. Peptide sequence homology with COX-2 and COX-2b was found for a length of 19 amino acids. Based on immunodetection, molecular weight and equivical MALDI-TOF results, one of these up-regulated proteins may be COX-2b. [source] Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresisELECTROPHORESIS, Issue 16 2003Gianfranco Mamone Abstract We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five ,-, fifteen ,s1 -, ten ,s2 -, and four ,-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to ,-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single ,-CN component. The phosphate group on site Ser12 of tryptic peptide 8,22 of most phosphorylated ,s1 -CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two ,-CN components was determined by means of MS/MS analysis. [source] Synthesis of Carrier-Transporting Dendrimers with Perylenebis(dicarboximide)s as a Luminescent CoreEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 4 2006Jianfeng Pan Abstract Well-defined, modular dendrimers enable processing techniques and electronic properties to be tuned independently. Moreover, the dendritic topology can isolate the core chromophore, thus reducing or eliminating strong intermolecular interactions. This paper presents the synthesis of three series of flexible, dendron-functionalized dendrimers as red-light-emitting materials by a convergent approach: (1) carbazole (CZ) or oxadiazole (OXZ) terminated imide-type dendrimers, (2) cascade energy-transferring imide-type dendrimers, and (3) CZ-terminated perylene bay-type dendrimers. They all consist of the luminescent core of perylenebis(dicarboximide)s with specific functional groups of CZ or OXZ at the periphery and are constructed from flexible Fréchet-type poly(aryl ether) dendrons. The chemical structures of the dendrons and dendrimers were determined by standard spectroscopic techniques including 1H and 13C NMR spectroscopy and low/high-resolution mass spectrometry (ESI or MALDI-TOF). The dendrimers are designed on the basis of the following considerations: (1) dendron functionalization to incorporate CZ or OXZ units to realize the carrier-injection adjustment, (2) tuning or improving solubility, functionality, glass-transition temperature (Tg) with well-defined dendrons, and (3) avoiding luminescence quenching with the help of high site-isolation of dendrons to enhance core luminescence. DSC results indicate that the incorporation of Fréchet-type poly(aryl ether) dendrons can improve the amorphous properties and increase Tg. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Towards a Selective Functionalization of Amino-Terminated DendrimersEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 22 2004Fritz Vögtle Abstract Selective functionalization of the periphery of commercial polypropyleneamine (POPAM) and polyamidoamine (PAMAM) dendrimers has been investigated in preparative scale. The first generation (G1) POPAM dendrimer was for the first time selectively N,N -bis(sulfonylated) with tosyl chloride and the corresponding mono-, di-, tri-, and tetra- N -tosylsulfonamides were isolated and fully characterized. Unexpectedly, similar persulfonylation of G2 POPAM results in splitting of a central C,N bond and only fully and partially sulfonylated halves of the initial dendrimer could be isolated. Higher generations of POPAM are also split during the persulfonylation yielding complex mixtures of persulfonylated dendritic fragments which could hardly be identified. A plausible mechanism of the POPAM decomposition on the basis of the reaction product analysis is proposed. N -Sulfonylation of a peripheral octasulfonamide of G2 POPAM with tosyl chloride also leads to the destruction of the dendrimer, while its N -alkylation with benzyl bromide proved to be not selective yielding a completely alkylated derivative. Unlike POPAM dendrimers, PAMAM dendrimers were shown to be more stable during their sulfonylation and no decomposition of the dendritic backbone was detected. In contrast to the POPAM dendrimers, PAMAM dendrimers were shown to be rather inert with respect to the formation of N -tosylsulfonamides since they could only be N -monosulfonylated at all peripheral amino groups. The combination of MALDI-TOF and ESI-FT-ICR tandem mass spectrometry has been shown to be an effective method for structure assignment and purity check of selectively or fully persulfonylated dendritic oligoamines. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [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] The identification of a phospholipase B precursor in human neutrophilsFEBS JOURNAL, Issue 1 2009Shengyuan Xu A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be , 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn -1 and sn -2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. [source] IPSE/alpha-1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated N-glycansFEBS JOURNAL, Issue 10 2006Manfred Wuhrer Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs. [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] 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] A Macrocyclic Model Dodecamer for Polyfluorenes,ADVANCED MATERIALS, Issue 1 2009Sascha C. Simon A , -conjugated, cyclododeca-2,7-fluorene macrocycle is synthesized as a new model compound for polyfluorenes. The structure formation is demonstrated by 1H NMR spectroscopy, MALDI-TOF, and STM measurements, and the optical properties of this monodisperse cycle are investigated using UV-vis and fluorescence spectroscopy. [source] Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrolJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2006Karel Bezstarosti Abstract Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardiopro-tection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apopto-sis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and precondi-tioning-related proteins were identified that were all upregulated by resveratrol: MAPKK, two different aB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxire-doxin-2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -aB-crys-tallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofila-ment regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins. [source] Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2010Song-Lin Shi Abstract In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK-N-SH cells pre- and post-treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two-dimensional gel electrophoresis and MALDI-TOF showed that NPM was a component of the nuclear matrix and its expression in SK-N-SH cells post-treated with RA was down-regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK-N-SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c-myc, c-fos, p53, and Rb in SK-N-SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK-N-SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67,74, 2010. © 2010 Wiley-Liss, Inc. [source] Modern MALDI time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009Marvin L. Vestal Abstract This paper focuses on development of time-of-flight (TOF) mass spectrometry in response to the invention of matrix-assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI-TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high-performance MALDI-TOF and TOF-TOF with off-line high-capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC-MS and MS-MS. Copyright © 2009 John Wiley & Sons, Ltd. [source] Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus speciesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001Gerold Bacher Abstract A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix ,-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8,10 pmol range and with a mass accuracy of ±0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2,4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2,4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MSn experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Copyright © 2001 John Wiley & Sons, Ltd. [source] Identification of three novel peptides isolated from the venom of the neotropical social wasp Polistes major majorJOURNAL OF PEPTIDE SCIENCE, Issue 7 2007Václav, ovský Abstract Three novel peptides designated as PMM1, PMM2, and PMM3 were isolated and characterized from the venom of the social wasp Polistes major major, one of the most common wasps in the Dominican Republic. By Edman degradation, and MALDI-TOF and ESI-QTOF mass spectrometry, the primary sequences of these peptides were established as follows: PMM1, H-Lys-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH (1357.77 Da); PMM2, H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-Ser-Ile-Gly-Lys-Glu-Val-Leu-Lys-Ala-Leu-NH2 (1909.19 Da); and PMM3, H-Phe-Leu-Ser-Ala-Leu-Leu-Gly-Met-Leu-Lys-Asn-Leu-NH2 (1317.78 Da). The suggested sequences were confirmed by MS analysis of peptide fragments obtained by enzymatic digestion. The peptide PMM1 is a lysyl-arginyl-Thr6 -bradykinine that belongs to the wasp kinins group. The sequence of the PMM2 peptide is unique; it resembles somewhat the tetradecapeptide amides of the mastoparan group; however, the chain is extended by three additional amino acid residues. The sequence of PMM3 dodecapeptide is homologous to the peptides of the wasp chemotactic group. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] 71 Proteomics of haematococcus pluvialis: new opportunities for study of genomics of a non-sequenced speciesJOURNAL OF PHYCOLOGY, Issue 2003Q. Hu The green alga, Haematococcus pluvialis, has become a model organism for commercial production of the high-value carotenoid astaxanthin. H. Pluvialis has also drawn significant scientific attention because fundamental biological questions relating to the massive cellular accumulation of astaxanthin have to be addressed in order to improve the yield and quality of the algal biomass. However, research has been impeded by the lack of molecular background information on this non-sequenced species. A combination of classical biochemistry with a state-of-the-art proteomic approach was used to address these questions. This was possible by taking advantage of information already available for homologous genes/gene-products in organisms whose genomes have been sequenced. The approach involved isolation of subsets of the proteome from subcellular compartments/organelles of an organism by one- or two-dimensional electrophoresis (1-DE or 2-DE) and their identification by N-terminal sequencing and peptide mass fingerprinting (PMF), involving matrix-assisted laser desorption/ionization and time-of-flight (MALDI-TOF) mass spectrometry coupled with bioinformatics. Based upon the information obtained from the combined methods, expression and physiological functions of specific genes/encoded proteins may be deduced. Examples include profiling of cell wall proteins, biogenesis and protein composition of lipid bodies, and expression patterns of soluble proteins under stress conditions. Advantages and limitations of the method for non-sequenced organisms and for cross-species protein identification will also be discussed. [source] Poly(arylene sulfide)s by nucleophilic aromatic substitution polymerization of 2,7-difluorothianthreneJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 9 2009Maxwell J. Robb Abstract Poly(thianthrene phenylene sulfide) and poly(thianthrene sulfide) have been prepared by nucleophilic aromatic substitution polymerization of the activated monomer 2,7-difluorothianthrene with bis thiophenoxide and sulfide nucleophiles, respectively. The resulting polymers are thermally stable, amorphous materials that have been characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), gel permeation chromatography (GPC), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, UV-Vis spectroscopy, refractometry, and intrinsic viscosity (IV) measurements. The polymers produced exhibit 5% weight loss values approaching 500 °C in inert and air atmospheres and glass transition temperatures that range from 149 to 210 °C. Poly(thianthrene phenylene sulfide) with a number average molecular weight of 22,100 g/mol has been synthesized with an IV in DMPU of 0.62 dL/g at 30 °C. Creasable films of this polymer have been prepared by solvent casting and melt pressing at 250 °C. Films of poly(thianthrene phenylene sulfide) exhibit transparencies greater than 50% at wavelengths exceeding 400 nm and a high refractive index value of 1.692 at a wavelength of 633 nm, making the polymer interesting for optical applications. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2453,2461, 2009 [source] Cyclic poly(pyridine ether)s by the polycondensation of 2,6-difluoropyridine with various diphenolsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 20 2005Hans R. Kricheldorf Abstract The bistrimethylsilyl derivatives of six different diphenols were polycondensed with 2,6-difluoropyridine in N -methylpyrrolidone in the presence of K2CO3. On the basis of previous studies, the reaction conditions were optimized for almost quantitative conversions. The feed ratio was systematically varied to optimize the molecular weight. A 2 mol % excess of 2,6-difluoropyridine was needed to obtain maximum molecular weights. In the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of the optimized polyethers, only cycles were found (detectable up to 5000 Da). Obviously, the relatively low molecular weights obtained under optimized conditions resulted from a limitation of the chain growth by cyclization, indicating a high cyclization tendency for poly(pyridine ether)s. The size exclusion chromatography measurements not only proved low molecular weights but also demonstrated the existence of bimodal mass distributions and high polydispersities. Protonation of the poly(pyridine ether)s required strong acids such as methane or trifluoromethane sulfonic acid. The solubilities of the neutral and protonated polyethers derived from bisphenol A were studied in various solvents. The MALDI-TOF mass spectra proved that protonation at 20,25 °C did not cause cleavage of ether bonds. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4781,4789, 2005 [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry study on copolymers obtained by the alternating copolymerization of bis(,-lactone) and epoxide with potassium tert -butoxideJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 12 2005Chenxi Zhang Abstract Oligomer samples obtained by the anionic copolymerization of a bis(,-lactone), 2,8-dioxa-1-methylbicyclo[3.3.0]octane-3,7-dione (1), and glycidyl phenyl ether with potassium tert -butoxide have been analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The MALDI-TOF mass spectra of these cooligomers show well-resolved signals that can be reliably assigned to linear, alternating cooligomers that have carboxylate chain ends or alkoxide chain ends and cyclic ones. The formation of these three series of cooligomers suggests that the polymerization process involves concomitant intermolecular transesterification and intramolecular back-biting. The intramolecular back-biting reaction causes the formation of cyclic cooligomers, whereas the intermolecular transesterification causes the reduction of the molecular weight and the transformation of the alkoxide active chain end into a carboxylate chain end. The MALDI-TOF mass spectrometry study has shown that an excess of monomer 1 enhances the selectivity of propagation by increasing the probability of the attack of the alkoxide chain end to 1. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2643,2649, 2005 [source] Synthesis and characterization of 2,2-bis(methylol)propionic acid dendrimers with different cores and terminal groupsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 7 2004Michael Malkoch Abstract Three sets of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) were synthesized. Two of the sets had benzylidene terminal groups and either a trimethylolpropane or triphenolic core moiety. The last set had acetonide terminal groups and a triphenolic core moiety. Benzylidene-[G#1]-anhydride and acetonide-[G#1]-anhydride were used as the reactive building blocks in the construction of all dendrimers. The large excess of building blocks used in the coupling reactions initially resulted in considerable material loss. This waste was eliminated through the development of a recycling method. 1H and 13C NMR and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis were used to verify the purity of all compounds. Size exclusion chromatography (SEC) was used, as well as MALDI-TOF, for molecular weight determinations. The SEC measurements were conducted with a universal calibration method and an online right-angle laser light scattering detector. Measured dendrimer molecular weights were close to their theoretical molar masses. Observations were also made of the hydrodynamic radius and intrinsic viscosity for the different dendrimers. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 1758,1767, 2004 [source] Radical Telomerisation of Vinyl Phosphonic Acid with a Series of Chain Transfer AgentsMACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 6 2007Ghislain David Abstract The use of CTAs for the radical polymerisation of VPA has been successfully applied for the first time. Several CTAs were investigated, including thiol, disulfide and halogenated compounds. The use of mercaptoacetic acid led to the synthesis of the mercaptan VPA monoadduct, which was attributed to a high transfer constant (CT,=,ktr/kp) of approximately 7. Transfer reactions with VPA appeared to be unsuccessful when using benzyldisulfide, 1,1,1-trichloroethane and trichloroacetic acid. Application of bromotrichloromethane as a transfer agent led to the oligomerisation of VPA, with final conversion being approximately 80%. The transfer events were characterised by 13C NMR, MALDI-TOF and elemental analysis. Molecular weights of the successful VPA polymerisations showed values ranging from 600 to 5,000 g,·,mol,1, which were in close agreement with the theoretically calculated values. [source] Genetic evidence for a role of IL33 in nasal polyposisALLERGY, Issue 5 2010I. D. Buysschaert To cite this article: Buysschaert ID, Grulois V, Eloy P, Jorissen M, Rombaux P, Bertrand B, Collet S, Bobic S, Vlaminck S, Hellings PW, Lambrechts D. Genetic evidence for a role of IL33 in nasal polyposis. Allergy 2010; 65: 616,622. Abstract Background:, Little is known about the genetic factors that contribute to nasal polyposis (NP). A genome-wide association study identified 10 single nucleotide polymorphisms (SNPs) associated with eosinophilia. As eosinophils play a key role in the pathogenesis of NP, we assessed if any of these SNPs contribute to genetic susceptibility of NP. Methods:, We recruited 284 patients with NP in four participating hospitals in Belgium and 427 healthy controls, and genotyped 10 SNPs affecting eosinophilia (rs1420101 in IL1RL1, rs12619285 in IKZF2, rs4431128 in GATA2, rs4143832 in IL5, rs3184504 in SH2B3, rs2416257 in WDR36, rs2269426 in MHC, rs9494145 in MYB, rs748065 in GFRA2, and rs3939286 in IL33) using MALDI-TOF. A two-stage design was used while correcting for multiple testing. Results:, First stage analysis, involving 150 NP patients and 250 controls, identified rs3939286 nearby IL33 as a susceptibility factor for NP. Per at-risk A-allele, rs3939286 increased the risk for NP with an odds ratio (OR) of 1.60 (95% CI = 1.16,2.22; P = 0.0041). Second stage replication analysis in another 123 NP patients and 165 controls confirmed this association (OR = 1.43; CI = 1.00,2.06; P = 0.046). The combined analysis of both stages revealed an OR of 1.53 (CI = 1.21,1.96; P = 0.00041). Given the association of IL33 with NP, we also investigated rs1420101 in IL1RL1, which is the receptor for IL33. Although rs1420101 itself failed to associate with NP, a combined risk assessment of rs3939286 and rs1420101 further increased the risk for NP. Conclusion:, We provide unprecedented genetic evidence suggesting a role for the IL33 pathway in the pathogenesis of NP. [source] Evidence that WbpD is an N -acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O-antigen biosynthesis in Pseudomonas aeruginosa PAO1MOLECULAR MICROBIOLOGY, Issue 5 2005Cory Q. Wenzel Summary Di- N -acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3- N -acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy- d -mannuronic acid [UDP- d -Man(2NAc3NAc)A], a precursor for the d -Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen (, 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthesis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (L,H) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N-terminally histidine-tagged fusion protein (His6,WbpD) and purified to >,95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left-handed helical structure, which substantiated in silico predictions made earlier. Results from SDS-PAGE, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6 -WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incubation of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure-based model for the L,H domain of WbpD was generated. Comparisons between this model and the L,H domains of known HexATs suggested that Lys136 plays a role in acetyl-CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP- d -Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP-2-acetamido-3-amino-2,3-dideoxy- d -glucuronic acid 3- N -acetyltransferase. [source] Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 4 2002A. R. Hesketh Summary The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their anno-tated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites. [source] Production and characterization of an allergen panel for component-resolved diagnosis of celery allergyMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue S2 2008Merima Bublin Abstract In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future. [source] Isolation and Characterisation of Procyanidins from Rumex obtusifoliusPHYTOCHEMICAL ANALYSIS, Issue 3 2007Paul Spencer Abstract An acetone:water (7:3) extract obtained from the leaves of Rumex obtusifolius was fractionated into procyanidin oligomer and polymer fractions using a linear gradient and a simple step method on Sephadex LH-20. The chemical characteristics of the procyanidin fractions were studied by 13C-NMR spectroscopy, acid-catalysed degradation in the presence of benzyl mercaptan, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS and electrospray ionisation (ESI) MS. The 13C-NMR showed that the polymer fraction consisted predominantly of procyanidin polymers, some with galloyl groups attached. The thiolysis reaction products indicated a mean degree of polymerisation (DP) of 4.3 for the step method, and a range of 2.3,8.2 mean DP for the gradient fractionation, with epicatechin as the most abundant flavan-3-ol extension unit, while the terminal units consisted of equal proportions of catechin, epicatechin and epicatechin gallate. Singly charged ions observed in MALDI-TOF/MS showed a range of oligomeric procyanidins and their polygalloyl derivatives. These species (in the range DP 2,7) were also observed by ESI/MS but the spectra were more complex due to overlapping multiply charged ions. Isolation of oligomers from the Sephadex LH-20 fraction by chromatography on polyamide and C18 yielded B1, B2, B3 and B7 dimers, an A-type trimer and a B2 3,3,- O -digallate. Copyright © 2007 John Wiley & Sons, Ltd. [source] Structural analysis of ,-Gal and new non-Gal carbohydrate epitopes from specific pathogen-free miniature pig kidneyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2008Yun-Gon Kim Abstract The major barrier in transplantation of pig organs into humans is the presence of surface carbohydrate antigens (e.g., the Gal,1-3Gal,1-4GlcNAc-R (,-Gal) epitope) expressed on pig endothelial cells. In this study, total N -glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney are identified by MALDI-TOF, negative ion ESI MS/MS and normal-phase HPLC (NP-HPLC) combined with exoglycosidase digestion. Over 100 N -glycans, including sialylated and neutral types, were identified. As well as the known ,-Gal antigens, some of these glycans contained novel non-Gal carbohydrate antigens such as (Neu5Gc-Gal-GlcNAc) and Gal,1-3Lewisx (Gal-Gal-(Fuc)GlcNAc) which have not been reported before in N -glycans from pig organs. The ability of MALDI, ESI, and HPLC to measure the relative proportions of the glycans was evaluated. The HPLC resolution was insufficient for accurate work and some minor differences were noted in the ionization efficiencies of different glycan groups when measured by the two mass spectrometric techniques. However, the results indicated that the relative quantity of ,-Gal epitope was in the region of 50% of the complex glycans. High-mannose type glycans were also abundant (35,43%) but appeared to be ionized more efficiently than the complex glycans by ESI than by MALDI. [source] Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2006Carlo Vascotto Abstract Knowledge of early molecular events occurring upon ischemia/reperfusion,(I/R) during liver transplantation,(LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R,injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon,LT. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF,MS, we identified 36,proteins which resulted significantly altered upon I/R,injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R,injury in liver. [source] dUTP Pyrophosphatase, its appearance in extracellular compartment may serve as a potential biomarker for N -methyl- N' -nitro- N -nitrosoguanidine exposure in mammalian cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2006Meiping Wu Abstract The monofunctional alkylating agent N -methyl- N' -nitro- N -nitrosoguanidine (MNNG) is a model chemical widely used for studying the molecular events induced by the widespread environmental N -nitroso alkylating carcinogen. Many studies have focused on understanding MNNG-induced mutagenesis and carcinogenesis. However, the search for specific indicators of MNNG exposure is still underway. In this study, we analyzed the proteins in culture medium of human amnion epithelial cells (FL,cells) exposed to MNNG by 2-DE followed by MALDI-TOF,MS, in the hope of finding a specific protein marker suitable for MNNG risk assessment. Image visualization and statistical analysis indicated that 12,spots appeared and 4,spots up-regulated after MNNG exposure. Most of them were identified by MS. These proteins include nuclear isoform of dUTP pyrophosphatase (DUT-N), phosphoglycerate mutase,1, heparan sulfate proteoglycan perlecan, etc., which are involved in multiple cellular functions. Interestingly, 2-DE and MS analyses of cell lysate exposed to MNNG revealed that DUT-N was down-regulated. The appearance of DUT-N in culture medium and its down-regulation in cell lysate was confirmed by Western blot. These data suggest that these proteins, especially DUT-N, could be used as candidate biomarkers for monitoring MNNG exposure. [source] A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2006Annamraju D. Sarma Abstract Total protein extract of Bradyrhizobium japonicum cultivated in HM media were resolved by 2-D PAGE using narrow range IPG strips. More than 1200,proteins were detected, of which nearly 500,proteins were analysed by MALDI-TOF and 310,spots were tentatively identified. The present study describes at the proteome level a significant number of metabolic pathways related to important cellular events in free-living B.,japonicum. A comparative analysis of proteomes of free-living and nodule residing bacteria revealed major differences and similarities between the two states. Proteins related to fatty acid, nucleic acid and cell surface synthesis were significantly higher in cultured cells. Nitrogen metabolism was more pronounced in bacteroids whereas carbon metabolism was similar in both states. Relative percentage of proteins related to global functions like protein synthesis, maturation & degradation and membrane transporters were similar in both forms, however, different proteins provided these functions in the two states. [source] | |||||||||||||||||||||||||||||||