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Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry (matrix-assisted + laser_ionization_time-of-flight_mass_spectrometry)
Selected AbstractsCranberry proanthocyanidins are cytotoxic to human cancer cells and sensitize platinum-resistant ovarian cancer cells to paraplatinPHYTOTHERAPY RESEARCH, Issue 8 2009Ajay P. Singh Abstract Polyphenolic extracts of the principal flavonoid classes present in cranberry were screened in vitro for cytotoxicity against solid tumor cells lines, identifying two fractions composed principally of proanthocyanidins (PACs) with potential anticancer activity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) analysis of the proanthocyanidins (PACs) fractions indicated the presence of A-type PACs with 1,4 linkages containing between 2,8 epicatechin units with a maximum of 1 epigallocatechin unit. PACs exhibited in vitro cytotoxicity against platinum-resistant human ovarian, neuroblastoma and prostate cancer cell lines (IC50 = 79,479 µg/mL) but were non-cytotoxic to lung fibroblast cells (IC50 > 1000 µg/ml). SKOV-3 ovarian cancer cells treated with PACs exhibited classic apoptotic changes. PACs acted synergistically with paraplatin in SKOV-3 cells. Pretreatment of SKOV-3 cells with PACs (106 µg/ml) resulted in a significant reduction of the paraplatin IC50 value. Similarly, in a BrdU incorporation assay, co-treatment of SKOV-3 cells with PACs and paraplatin revealed reduced cell proliferation at lower concentrations than with either individually. In SKOV-3 cell cultures co-treated with PAC-1 and paraplatin, an HPLC analysis indicated differential quantitative presence of various PAC oligomers such as DP-8, -9, -11 and -14 indicating either selective binding or uptake. Cranberry proanthocyanidins exhibit cell-line specific cytotoxicity, induce apoptotic markers and augment cytotoxicity of paraplatin in platinum-resistant SKOV-3 ovarian cancer cells. Copyright © 2009 John Wiley & Sons, Ltd. [source] Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver,HEPATOLOGY, Issue 4 2009Anne T. Nies An important function of hepatocytes is the biotransformation and elimination of various drugs, many of which are organic cations and are taken up by organic cation transporters (OCTs) of the solute carrier family 22 (SLC22). Because interindividual variability of OCT expression may affect response to cationic drugs such as metformin, we systematically investigated genetic and nongenetic factors of OCT1/SLC22A1 and OCT3/SLC22A3 expression in human liver. OCT1 and OCT3 expression (messenger RNA [mRNA], protein) was analyzed in liver tissue samples from 150 Caucasian subjects. Hepatic OCTs were localized by way of immunofluorescence microscopy. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and genome-wide single-nucleotide polymorphism microarray technology served to genotype 92 variants in the SLC22A1-A3/OCT1-3 gene cluster. Transport of metformin by recombinant human OCT1 and OCT3 was compared using transfected cells. OCT1 mRNA and protein expression varied 113- and 83-fold, respectively; OCT3 mRNA expression varied 27-fold. OCT1 transcript levels were on average 15-fold higher compared with OCT3. We localized the OCT3 protein to the basolateral hepatocyte membrane and identified metformin as an OCT3 substrate. OCT1 and OCT3 expression are independent of age and sex but were significantly reduced in liver donors diagnosed as cholestatic (P , 0.01). Several haplotypes for OCT1 and OCT3 were identified. Multivariate analysis adjusted for multiple testing showed that only the OCT1-Arg61Cys variant (rs12208357) strongly correlated with decreased OCT1 protein expression (P < 0.0001), and four variants in OCT3 (rs2292334, rs2048327, rs1810126, rs3088442) were associated with reduced OCT3 mRNA levels (P = 0.03). Conclusion: We identified cholestasis and genetic variants as critical determinants for considerable interindividual variability of hepatic OCT1 and OCT3 expression. This indicates consequences for hepatic elimination of and response to OCT substrates such as metformin. (HEPATOLOGY 2009.) [source] Anionic polymerization of methyl methacrylate initiated with late transition-metal halides/organolithium/triisobutylaluminum systemsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 13 2003Eiji Ihara Abstract Anionic polymerization of methyl methacrylate (MMA) initiated with late transition-metal halides [manganese chloride (MnCl2), iron dichloride (FeCl2), iron trichloride (FeCl3), cobalt chloride (CoCl2), or nickel bromide (NiBr2)]/organolithium [nButyllithium (nBuLi) or phenyllithium (PhLi)]/triisobutylaluminum (iBu3Al) systems is described. Except for the system with NiBr2, the polymerizations of MMA afforded narrow molecular weight distribution poly(methyl methacrylate)s (PMMAs) with high molecular weights in quantitative yields at 0 °C in toluene. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of the PMMAs obtained by the systems with FeCl2, FeCl3, and CoCl2 revealed that the polymers had hydrogen (H) at both chain ends. Accordingly, the reaction of the transition-metal halides with the organolithium in the presence of iBu3Al should result in the formation of transition-metal hydride [M-H], species, which was nucleophilic enough to initiate the MMA polymerization. Because the presence of a six-membered cyclic structure resulting from backbiting was confirmed from the MALDI-TOF MS analyses of the PMMA obtained with the metal halide (FeCl2, FeCl3, or CoCl2)/organolithium systems in the absence of iBu3Al, the introduction of H at the ,-chain end indicated that iBu3Al should prevent the backbiting. However, the MnCl2/nBuLi/iBu3Al initiating system gave PMMAs bearing H at the , chain end and six-membered cyclic structure at the , chain end. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1962,1977, 2003 [source] Matrix/analyte ratio influencing polymer molecular weight distribution in matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2009Gitta Schlosser Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been used to characterize poly(L-lysine) polymers and unique oligomer peptides, like 10-, 15- and 25-mer [Lys]n oligolysine peptides. Several matrices have been tried in order to find optimal conditions, but only , -cyano-4-hydroxycinnamic acid gave analytically useful spectra. The synthetic oligomers and their mixtures gave good quality spectra, showing protonated and cationized molecules, including doubly charged species. The polymers, analogously, gave a wide distribution of single- and double-cationized peak series. The polymer distributions observed indicate the presence of significant suppression effects. The concentration (matrix/analyte ratio) was found to influence the results significantly; distributions shifting to higher masses when higher polymer concentrations were used. This effect was studied in detail using the synthetic (,monodisperse') oligolysine peptides. It was found that the relative intensities change by over an order of magnitude in the 0.1,10,pmol/µL concentration range (typical for most proteomic analyses). The results indicate that concentration effects need to be considered when MALDI-MS is used for quantitative purposes. Copyright © 2009 John Wiley & Sons, Ltd. [source] Characterization of different poly(2-ethyl-2-oxazoline)s via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2009Anja Baumgaertel Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with CID (collision-induced dissociation) has been used for the detailed characterization of two poly(2-ethyl-2-oxazoline)s as part of a continuing study of synthetic polymers by MALDI-TOF MS/MS. These experiments provided information about the variety of fragmentation pathways for poly(oxazoline)s. It was possible to show that, in addition to the eliminations of small molecules, like ethene and hydrogen, the McLafferty rearrangement is also a possible fragmentation route. A library of fragmentation pathways for synthetic polymers was also constructed and such a library should enable the fast and automated data analysis of polymers in the future. Copyright © 2009 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChipÔ sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrateRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2005Leon P. Oehlers The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChipÔ. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to , -cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50,mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase , tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Copyright © 2005 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a tool for fast identification of protein binders in color layers of paintingsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2004Radovan Hynek Identification of materials in color layers of paintings is necessary for correct decisions concerning restoration procedures as well as proving the authenticity of the painting. The proteins are usually important components of the painting layers. In this paper it has been demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be used for fast and reliable identification of proteins in color layers even in old, highly aged matrices. The digestion can be easily performed directly on silica wafers which are routinely used for infrared analysis. The amount of material necessary for such an analysis is extremely small. Peptide mass mapping using digestion with trypsin followed by MALDI-TOFMS and identification of the protein was successfully used for determination of the binder from a painting of the 19th century. Copyright © 2004 John Wiley & Sons, Ltd. [source] Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial culturesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2002Torsten Stein Bacillus subtilis synthesizes the lanthionine containing 32-amino-acid peptide antibiotic (lanti-biotic) subtilin from a ribosomally generated 56-amino-acid precursor pre-propeptide by extensive posttranslational modifications. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to monitor the production of matured subtilin within crude samples taken from B. subtilis culture media without prior fractionation. The processing reaction of subtilin was blocked with the serine protease inhibitor phenylmethylsulfonyl fluoride and different subtilin precursor peptides in the molecular mass range up to 6220 were observed. Two of these species were isolated by reversed-phase high-performance liquid chromatography (HPLC) and structurally analyzed by post-source decay MALDI-TOFMS. We provide evidence that the precursor species comprise the posttranslational modified C-terminal part of subtilin to which leader peptide moieties with different chain lengths are attached. These antimicrobial-inactive species could be processed to antibiotic-active subtilin by incubation with culture media of different subtilin-nonproducing B. subtilis strains as indicated by a combination of antimicrobial growth assays and MALDI-TOFMS analyses. These achievements are strong evidence for the sensitivity of MALDI-TOFMS methodology that allows straightforward investigations of analytes even in complex mixtures without time-consuming sample preparations. Copyright © 2001 John Wiley & Sons, Ltd. [source] Characterization of volatile compounds and triacylglycerol profiles of nut oils using SPME-GC-MS and MALDI-TOF-MSEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 2 2009Stefanie Bail Abstract Several nut oil varieties mainly used as culinary and overall healthy food ingredients were subject of the present study. Headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was employed in order to determine the qualitative composition of volatile compounds. Furthermore, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used in order to assess the profiles and relative composition of the prevalent triacylglycerols (TAG) within the oils. The headspace of the majority of oil samples was dominated by high contents of acetic acid (up to 42%) and hexanal (up to 32%). As nut oils are typically gained by cold-pressing from previously roasted nuts, characteristic pyrazine derivatives as well as degradation products of long-chain fatty acids were detected. TAG analysis of these oils revealed a quite homogeneous composition dominated by components of the C52 and C54 group composed mainly of oleic (18:1), linoleic (18:2), stearic (18:0) and palmitic (16:0) acid residues representing together between 65 and 95% of the investigated nut oils. The TAG profiles showed characteristic patterns which can be used as ,fingerprints' of the genuine oils. Nut oils exhibiting quite similar fatty acid composition (e.g. hazelnut, pistachio and beech oil) could be clearly discriminated based on TAG showing significant differences between the oils. [source] Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cellsEXPERIMENTAL DERMATOLOGY, Issue 9 2004Moon Kyung Ha Abstract:, Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3,10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14,15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging. [source] Comparative proteomic analysis of passaged Helicobacter pyloriJOURNAL OF BASIC MICROBIOLOGY, Issue 5 2009Mao-Jun Zhang Abstract In order to identify the proteins associated with Helicobacter pylori colonization in mice, we used 2-dimensional gel electrophoresis (2-DE) to analyze the membrane- and soluble-cellular proteins extracted from H. pylori strain 26695 and the mouse-passaged homolog 88-3887. We defined 2- and 3-fold changes in protein expression as the threshold values for differential expression in the membrane-protein and whole-cell-protein fractions, respectively. The differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). A total of 29 proteins, including 16 membrane- or membrane-associated proteins (13 upregulated, 3 downregulated) and 13 cellular proteins (10 upregulated, 3 downregulated) were differentially expressed between the strains 26695 and 88-3887. Among the upregulated proteins, 10 proteins had been previously shown to be associated with the mouse colonization, and 13 upregulated proteins were shown to be associated with the adaptation of H. pylori in murine hosts for the first time in this study. The identified proteins were classified as proteins related to metabolism, stress response, virulence, or adhesion. The data presented in this report indicated that there were subsets of upregulated proteins in mouse-adapted H. pylori. In particular, the adhesins, virulence factors, and stress-response proteins are likely to contribute to colonization in mice. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Role of estrogenic compounds (diethylstibestrol, 17,-estradiol, and bisphenol A) in the phosphorylation of substrate by protein kinase C,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2009Jeong-Hun Kang Abstract Estrogenic compounds can activate protein kinase C (PKC), which is a calcium and phospholipid-dependent serine/threonine kinase. In the present study, we investigated the role of 17,-estradiol (E2), diethylstibestrol (DES), and bisphenol A (BPA) in the phosphorylation of substrate by PKC, using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The level of phosphorylated peptide was low in the absence of phosphatidylserine (PS). Moreover, reduction of phosphorylation ratios was identified in the presence of diacylglycerol (DAG) and Ca2+ or PS and Ca2+ after adding E2, DES, and BPA. However, no change in phosphorylation ratios was found in the presence of DAG and PS. Addition of E2, DES, and BPA also had no influence on the phosphorylation reaction of substrate by cell or tissue lysate samples. Our study suggests that E2, DES, and BPA can bind to the C2 domain of PKC, but have no effects on the phosphorylation reaction of substrates in the presence of DAG and PS. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:318,323, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20294 [source] Proteomic Alterations of Antarctic Ice Microalga Chlamydomonas sp.JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 8 2006Under Low-Temperature Stress Abstract Antarctic ice microalga can survive and thrive in cold channels or pores in the Antarctic ice layer. In order to understand the adaptive mechanisms to low temperature, in the present study we compared two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of normal and low temperature-stressed Antarctic ice microalga Chlamydomonas sp. cells. In addition, new protein spots induced by low temperature were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both normal and low temperature-stressed cells were acquired. A total of 626 spots was detected in control cells and 652 spots were detected in the corresponding low temperature-stressed cells. A total of 598 spots was matched between normal and stressed cells. Two newly synthesized proteins (a and b) in low temperature-stressed cells were characterized. Protein spot A (53 kDa, pI 6.0) was similar to isopropylmalate/homocitrate/citramalate synthases, which act in the transport and metabolism of amino acids. Protein spot b (25 kDa, pI 8.0) was related to glutathione S -transferase, which functions as a scavenger of active oxygen, free radicals, and noxious metabolites. The present study is valuable for the application of ice microalgae, establishing an ice microalga Chlamydomonas sp. proteome database, and screening molecular biomarkers for further studies. (Managing editor: Li-Hui Zhao) [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] Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2003Lyuben N. Marekov Abstract High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of ,peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed. Copyright © 2003 John Wiley & Sons, Ltd. [source] Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibrationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2002Omar Belgacem Abstract Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de- N -glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of ±0.12 and ±0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip® technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III, and AT-III,, were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de- N -glycosylation (by means of PNGase F) of the ,- and ,-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (,0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N - and O -glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous ,-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N -glycans. Copyright © 2002 John Wiley & Sons, Ltd. [source] In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2002S. Kilz Abstract Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide brigded, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF,pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O -glycosylated proteins up to 150 kDa after SDS-PAGE separation. Copyright © 2002 John Wiley & Sons, Ltd. [source] The different forms of PNS myelin P0 protein within and outside lipid raftsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Anna Fasano Abstract It is now well established that plasma membranes, such as the myelin sheath, are made of different microdomains with different lipid and protein composition. Lipid rafts are made mainly of sphingolipids and cholesterol, whereas the non-raft regions are made mainly of phosphoglycerides. Most myelin proteins may distribute themselves in raft and non-raft microdomains but the driving force that gives rise to their different distribution is not known yet. In this paper, we have studied the distribution of protein zero (P0), the most representative protein of PNS myelin, in the membrane microdomains. To this end, we have purified P0 from both non-raft (soluble P0, P0-S) and raft (P0-R) regions of PNS. Purified proteins were analyzed by two-dimensional gel electrophoresis and identified and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A detailed structural description of the two P0 forms is given in terms of amino acid sequence, post-translational modifications, and composition of associated lipids. Our findings suggest that structural differences between the two proteins, mainly related to the glycogroups, might be responsible for their different localization. [source] PURIFICATION AND CHARACTERIZATION OF A LECTIN, BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) ,JOURNAL OF PHYCOLOGY, Issue 1 2006Gwang Hoon Kim When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin,carbohydrate complementary system. Two sugars, N -acetyl- d -glucosamine and N -acetyl- d -galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate-labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N -acetyl- d -galactosamine and N -acetyl- d -glucosamine in the crude extract. One-step column purification using N -acetyl- d -glucosamine-agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross-linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-terminal 15 amino acid sequence of the lectin was Ser,Asp,Leu,Pro,Thr,X,Asp,Phe,Phe,His,Ile,Pro,Glu,Arg,Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa"bryohealin." [source] Cyclic alkoxyamine-initiator tethered by azide/alkyne-"click"-chemistry enabling ring-expansion vinyl polymerization providing macrocyclic polymersJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 15 2010Atsushi Narumi Abstract A cyclic initiator for the nitroxide-mediated controlled radical polymerization (NMP) is a powerful tool for the preparation of macrocyclic polymers via a ring-expansion vinyl polymerization mechanism. For this purpose, we prepared a Hawker-type NMP-initiator that includes an azide and a terminal alkyne as an acyclic precursor, which is subsequently tethered via an intramolecular azide/alkyne-"click"-reaction, producing the final cyclic NMP-initiator. The polymerization reactions of styrene with cyclic initiator were demonstrated and the resultant polymers were characterized by the gel permeation chromatography (GPC) and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These results prove that the ring-expansion polymerization of styrene occurred together with the radical ring-crossover reactions originating from the exchange of the inherent nitroxides generating macrocyclic polystyrenes with higher expanded rings. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3402,3416, 2010 [source] Determination of block size in poly(ethylene oxide)- b -polystyrene block copolymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 13 2009Marion Girod Abstract Characterization of block size in poly(ethylene oxide)- b -poly(styrene) (PEO- b -PS) block copolymers could be achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after scission of the macromolecules into their constituent blocks. The performed hydrolytic cleavage was demonstrated to specifically occur on the targeted ester function in the junction group, yielding two homopolymers consisting of the constitutive initial blocks. This approach allows the use of well-established MALDI protocols for a complete copolymer characterization while circumventing difficulties inherent to amphiphilic macromolecule ionization. Although the labile end-group in PS homopolymer was modified by the MALDI process, PS block size could be determined from MS data since polymer chains were shown to remain intact during ionization. This methodology has been validated for a PEO- b -PS sample series, with two PEO of number average molecular weight (Mn) of 2000 and 5000 g mol,1 and Mn(PS) ranging from 4000 to 21,000 g mol,1. Weight average molecular weight (Mw), and thus polydispersity index, could also be reached for each segment and were consistent with values obtained by size exclusion chromatography. This approach is particularly valuable in the case of amphiphilic copolymers for which Mn values as determined by liquid state nuclear magnetic resonance might be affected by micelle formation. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3380,3390, 2009 [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry investigations of polystyrene and poly(methyl methacrylate) produced by monoacylphosphine oxide photoinitiationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 11 2007Faith J. Wyzgoski Abstract The chain-end-group composition was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of low-molecular-weight polystyrene (PS) and poly(methyl methacrylate) (PMMA) produced by free-radical polymerization with a monoacylphosphine oxide, (2,4,6-trimethylbenzoyl) diphenylphosphine oxide (TPO), as a photoinitiator. Gel permeation chromatography (GPC) fractionation of the PS and PMMA samples with initial polydispersities of 1.81 and 2.81, respectively, yielded improved MALDI-TOF MS spectra. Spectral analyses of the PS fractions showed distributions attributable to PS having two diphenylphosphinyl ends and PS having one diphenylphosphinyl end and/or one 2,4,6-trimethylbenzoyl end, indicating that a combination of PS radicals with the highly reactive diphenylphosphine oxide group at one end of the chains was the predominant mode of termination. MALDI-TOF MS results for PMMA fractions provided evidence for termination primarily by disproportionation, but structure determination was confounded by the presence of isobaric peaks. Discernible peaks were obtained by MALDI-TOF MS analyses of GPC fractions of TPO-initiated poly(methyl- d3 methacrylate- d5), in which the major product was PMMA with a diphenylphosphinyl end group and an abstracted deuterium end group, whereas the minor combination product had two diphenylphosphinyl chain ends. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 2161,2171, 2007 [source] Synthesis of amphiphilic copolymer brushes: Poly(ethylene oxide)-graft-polystyreneJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 15 2006Zhongyu Li Abstract A well-defined amphiphilic copolymer brush with poly(ethylene oxide) as the main chain and polystyrene as the side chain was successfully prepared by a combination of anionic polymerization and atom transfer radical polymerization (ATRP). The glycidol was first protected by ethyl vinyl ether to form 2,3-epoxypropyl-1-ethoxyethyl ether and then copolymerized with ethylene oxide by the initiation of a mixture of diphenylmethylpotassium and triethylene glycol to give the well-defined polymer poly(ethylene oxide- co -2,3-epoxypropyl-1-ethoxyethyl ether); the latter was hydrolyzed under acidic conditions, and then the recovered copolymer of ethylene oxide and glycidol {poly(ethylene oxide- co -glycidol) [poly(EO- co -Gly)]} with multiple pending hydroxymethyl groups was esterified with 2-bromoisobutyryl bromide to produce the macro-ATRP initiator [poly(EO- co -Gly)(ATRP). The latter was used to initiate the polymerization of styrene to form the amphiphilic copolymer brushes. The object products and intermediates were characterized with 1H NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Fourier transform infrared, and size exclusion chromatography in detail. In all cases, the molecular weight distribution of the copolymer brushes was rather narrow (weight-average molecular weight/number-average molecular weight < 1.2), and the linear dependence of ln[M0]/[M] (where [M0] is the initial monomer concentration and [M] is the monomer concentration at a certain time) on time demonstrated that the styrene polymerization was well controlled. This method has universal significance for the preparation of copolymer brushes with hydrophilic poly(ethylene oxide) as the main chain. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4361,4371, 2006 [source] One-pot synthesis of star-shaped aliphatic polyesters with hyperbranched cores and their characterization with size exclusion chromatographyJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 13 2006Tadeusz Biela Star-shaped aliphatic polyesters with hyperbranched cores were prepared by a two-step synthesis with the one-pot, arms-first core method. First, the polymerization of ,-caprolactone (CL) or L,L -lactide (LA) was initiated with aluminum isopropoxide trimer. Then, the resulting poly(CL),OAl< or poly(LA),OAl< living chains were employed as initiators for 5,5,-bis(oxepan-2-one) or 1,6-dioxaspiro[4,4]nonane-2,7-dione) polymerization. A sequence of chain growth and branching reactions led to the formation of starlike macromolecules. The progress of the polymerization was followed with size exclusion chromatography, and the products of the model reaction were also analyzed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. [source] Preparation of novel macromonomers and study of their polymerizationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 15 2004Guohua Deng Abstract Novel macromonomers of polystyrene and poly(tert -butyl acrylate) containing a methacryloyl group as a polymerizable unit and two chains of the same length were prepared in two steps: the synthesis of the precursors through the atom transfer radical polymerization of styrene and tert -butyl acrylate initiated by 1-hydroxymethyl-1,1-di[(2-bromoisobutyryloxy)methyl] ethane and the esterification of the hydroxyl group in the precursors with methacryloyl chloride. The molecular weight and polydispersity of the macromonomers were controllable because of the living nature of the atom transfer radical polymerization. Gel permeation chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and hydrolysis confirmed the structure of the novel macromonomers. The homopolymerization and copolymerization of the macromonomers were investigated to prepare branched copolymers in which two chains were grafted from every repeating unit. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 3887,3896, 2004 [source] Synthesis and properties of novel polyimides derived from 2,2,,3,3,-benzophenonetetracarboxylic dianhydrideJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 9 2004Xing-Zhong Fang Abstract A new synthetic route to 2,2,,3,3,-BTDA (where BTDA is benzophenonetetracarboxylic dianhydride), an isomer of 2,3,,3,,4,-BTDA and 3,3,,4,4,-BTDA, is described. Single-crystal X-ray diffraction analysis of 2,2,,3,3,-BTDA has shown that this dianhydride has a bent and noncoplanar structure. The polymerizations of 2,2,,3,3,-BTDA with 4,4,-oxydianiline (ODA) and 4,4,-bis(4-aminophenoxy)benzene (TPEQ) have been investigated with a conventional two-step process. A trend of cyclic oligomers forming in the reaction of 2,2,,3,3,-BTDA and ODA has been found and characterized with IR, NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and elemental analyses. Films based on 2,2,,3,3,-BTDA/TPEQ can only be obtained from corresponding polyimide (PI) solutions prepared by chemical imidization because those from their polyamic acids by thermal imidization are brittle. PIs from 2,2,,3,3,-BTDA have lower inherent viscosities and worse thermal and mechanical properties than the corresponding 2,3,,3,,4,-BTDA- and 3,3,,4,4,-BTDA-based PIs. PIs from 2,2,,3,3,-BTDA and 2,3,,3,,4,-BTDA are amorphous, whereas those from 3,3,,4,4,-BTDA have some crystallinity, according to wide-angle X-ray diffraction. Furthermore, PIs from 2,2,,3,3,-BTDA have better solubility, higher glass-transition temperatures, and higher melt viscosity than those from 2,3,,3,,4,-BTDA and 3,3,,4,4,-BTDA. Model compounds have been prepared to explain the order of the glass-transition temperatures found in the isomeric PI series. The isomer effects on the PI properties are discussed. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 2130,2144, 2004 [source] Phthalide as an activating group for the synthesis of poly(aryl ether phthalide)s by nucleophilic aromatic substitutionJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 17 2002Daniel M. Knauss Abstract The phthalide ring was examined as an activating group for nucleophilic aromatic substitution. The proposed mechanism by which activation occurs is through a ring opening of the phthalide ring to form a Meisenheimer-like , complex. 3,3-Bis(4-fluorophenyl)phthalide was synthesized and examined under different reaction conditions to determine its suitability for polymer formation. Semiempirical calculations at the PM3 level suggested that 3,3-bis(4-fluorophenyl)phthalide is only moderately activated, whereas 1H, 13C, and 19F NMR spectroscopy suggested that the monomer was not sufficiently activated for nucleophilic aromatic substitution. However, low-molecular-weight polymers (number-average molecular weight < 7000 g/mol) were produced from bisphenol A, hydroquinone, and phenolphthalein. The polymers were characterized by gel permeation chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, NMR spectroscopy, and differential scanning calorimetry. The polymers displayed relatively high glass-transition temperatures. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3046,3054, 2002 [source] Synthesis of multicyclic and grafted polystyrenesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 16 2001Bénédicte Lepoittevin Abstract Well-defined multicyclic polystyrenes are prepared in two steps. The first step is the preparation of a cyclic difunctional polystyrene by the reaction of ,,,-dilithiopolystyrene chains with 1,3-bis(phenylethenyl)benzene. Then, this product is covalently grafted to poly(chloromethylstyrene) chains leading to the formation of a high molar mass product containing linear and cyclic parts. As a model reaction and to optimize the previous reaction, a study of coupling of the linear difunctional model polystyrene with poly(chloromethylstyrene) is performed leading to grafted polystyrene. The grafted products are analyzed by size-exclusion chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and liquid chromatography at the exclusion-adsorption transition point. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2723,2730, 2001 [source] Characterization of New Amphiphilic Block Copolymers of N -Vinyl Pyrrolidone and Vinyl Acetate, 1 , Analysis of Copolymer Composition, End Groups, Molar Masses and Molar Mass Distributions,MACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 8 2010Nick Fandrich Abstract New amphiphilic block copolymers consisting of N -vinyl pyrrolidone and vinyl acetate were synthesized via controlled radical polymerization using a reversible addition/fragmentation chain transfer (RAFT)/macromolecular design via the interchange of xanthates (MADIX) system. The synthesis was carried out in 1,4-dioxane as process solvent. In order to get conclusions on the mechanism of the polymerization the molecular structure of formed copolymers was analysed by means of different analytical techniques. 13C NMR spectroscopy was used for the determination of the monomer ratios. End groups were analysed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This technique was also used to determine possible fragmentations of the RAFT end groups. By means of a combination of size exclusion chromatography, 13C NMR and static light scattering molar mass distributions and absolute molar masses could be analysed. The results clearly show a non-ideal RAFT mechanism. [source] Systematic MALDI-TOF CID Investigation on Different Substituted mPEG 2000MACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 6 2010Katrin Knop Abstract Methoxy poly(ethylene glycol) 2000 (mPEG 2000) samples were substituted via esterification reactions to convert the hydroxyl end group of the mPEG into potential initiators for the cationic ring opening polymerization of 2-oxazolines. These substitution products were investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); in addition, detailed MALDI-TOF/TOF-collision induced dissociation (CID) MS studies were performed to introduce this method as complementary structural characterization tool allowing the detailed analysis of the prepared macromolecules. The CID of the macroinitiators revealed 1,4-hydrogen and 1,4-ethylene eliminations forming very regular fragmentation patterns which showed, depending on the end groups, different fragmentation series. Furthermore, very pronounced McLafferty,+,1 rearrangements (1,5 hydrogen-transfer) of the introduced ester end groups were observed leaving the mPEG molecule as neutral acid. This incisive loss revealed the exact molar mass for each end group and, therefore, represents an important tool for end group determination of functionalized polymers. [source] | |||||||||||||||||||