Home About us Contact
|
Home About us Contact | ||
|
|
||
Desorption/ionization Time-of-flight (ionization + time-of-flight)
Kinds of Desorption/ionization Time-of-flight Terms modified by Desorption/ionization Time-of-flight Selected AbstractsFMRFamide gene and peptide expression during central nervous system development of the cephalopod mollusk, Idiosepius notoidesEVOLUTION AND DEVELOPMENT, Issue 2 2010Tim Wollesen SUMMARY Mollusks are a showcase of brain evolution represented by several classes with a varying degree of nervous system centralization. Cellular and molecular processes involved in the evolution of the highly complex cephalopod brain from a simple, monoplacophoran-like ancestor are still obscure and homologies on the cellular level are poorly established. FMRFamide (Phe-Ile-Arg-Phe-NH2)-related peptides (FaRPs) constitute an evolutionarily conserved and diverse group of neuropeptides in the central nervous system (CNS) of many metazoans. Herein, we provide a detailed description of the developing FMRFamide-like immunoreactive (Fa-lir) CNS of the pygmy squid Idiosepius notoides using gene expression analyses and immunocytochemistry. The open reading frame of the I. notoides FMRFamide gene InFMRF predicts one copy each of FIRFamide, FLRFamide (Phe-Leu-Arg-Phe-NH2), ALSGDAFLRFamide (Ala-Leu-Ser-Gly-Asp-Ala-Phe-Leu-Arg-Phe-NH2), and 11 copies of FMRFamide. Applying matrix-assisted laser desorption/ionization time-of-flight (ToF) mass spectrometry-based peptide profiling, we characterized all predicted FaRPs except ALSGDAFLRFamide. Two cell clusters express InFMRF and show FMRFamide-like-immunoreactivity within the palliovisceral ganglia, that is, the future posterior subesophageal mass, during the lobe differentiation phase. They project neurites via ventral axonal tracts, which form the scaffold of the future subesophageal mass. In the supraesophageal mass, InFMRF is first expressed during mid-embryogenesis in the superior and inferior buccal lobes. A neurite of the peduncle commissure represents the first Fa-lir element. Later, the sub- and supraesophageal mass interconnect via Fa-lir neurites and more brain lobes express InFMRF and FMRFamide-like peptides. InFMRF expression was observed in fewer brain lobes than Fa-lir elements. The early expression of InFMRF and FMRFamide-lir peptides in the visceral system and not the remaining CNS of the cephalopod I. notoides resembles the condition found in the majority of investigated gastropods. [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] Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2004Martin Zehl Abstract A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H]2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright © 2004 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] 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] Mixed iridium(III) and ruthenium(II) polypyridyl complexes containing poly(,-caprolactone)-bipyridine macroligandsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 17 2004Veronica Marin Abstract A hydroxy-functionalized bipyridine ligand was polymerized with ,-caprolactone utilizing the controlled ring-opening polymerization of ,-caprolactone in the presence of stannous octoate. The resulting poly(,-caprolactone)-containing bipyridine was characterized by 1H NMR and IR spectroscopy, and gel permeation chromatography, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, revealing the successful incorporation of the bipyridine ligand into the polymer chain. Coordination to iridium(III) and ruthenium(II) precursor complexes yielded two macroligand complexes, which were characterized by NMR, gel permeation chromatography, matrix-assisted laser desorption/ionization time-of-flight MS, cyclic voltammetry, and differential scanning calorimetry. In addition, both photophysical and electrochemical properties of the metal-containing polymers proved the formation of a trisruthenium(II) and a trisiridium(III) polypyridyl species, respectively. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4153,4160, 2004 [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] Study of human neutrophil peptides in saliva by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2009Ming-Hui Yang Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides , HNP 1, 2, and 3 , in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI-TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva-absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age-dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging. Copyright © 2009 John Wiley & Sons, Ltd. [source] Characterization by mass spectrometry of an unknown polysiloxane sample used under uncontrolled medical conditions for cosmetic surgeryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2008Cédric Schneider For a complete understanding of the raw material used for cosmetic surgery under uncontrolled medical conditions, an unknown sample of polydimethylsiloxanes has been investigated utilizing a combination of analytical techniques: pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS), electrospray ionization (ESI)-MS, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS, and liquid chromatography (LC)/MS. Among these techniques, the LC/APCI-MS coupling allowed the fastest and more effective analysis. In addition, the complexity of the mass spectra deduced from these LC/MS experiments was simplified compared to the mass spectra obtained by MALDI-TOF. In this work, we have demonstrated how the LC/APCI-MS coupling applied to polydimethylsiloxane samples permits the full characterization of samples where end groups of different nature can be present in very small quantities. Copyright © 2008 John Wiley & Sons, Ltd. [source] Applications of silver nanoparticles capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight and atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for rapid analysis of sulfur drugs and biothiols in human urineRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2008Kamlesh Shrivas A strategy is presented for the analysis of sulfur drugs and biothiols using silver nanoparticles (AgNPs) capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and atmospheric pressure-matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-ITMS). Biothiols adsorbed on the surface of AgNPs through covalent bonding were subjected to ultraviolet (UV) radiation that enabled desorption and ionization due to the excellent photochemical property of NPs. The proposed method has been successfully applied for the determination of cysteine and homocysteine in human urine samples using an internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for cysteine and homocysteine in urine sample are 7 and 22,nM, respectively, with a relative standard deviation (RSD) of <10%. The advantages of the present method compared with the methods reported in the literature for biothiol analysis are simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Additionally, we also found that the bare AgNPs can be directly used as the matrix in MALDI-TOF MS for the analysis of sulfur drugs without the addition of an extra proton source. Copyright © 2008 John Wiley & Sons, Ltd. [source] Negative ion dissociation of peptides containing hydroxyl side chainsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2008Dan Pu The dissociation of deprotonated peptides containing hydroxyl side chains was studied by electrospray ionization coupled with Fourier transform ion cyclotron resonance (ESI-FTICR) via sustained off-resonance irradiation collision induced dissociation (SORI-CID). Dissociation under post-source decay (PSD) conditions was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). This work included hexapeptides with one residue of serine, threonine, or tyrosine and five inert alanine residues. During SORI-CID and PSD, dissociation of [M,H], yielded c- and y-ions. Side-chain losses of formaldehyde (HCHO) from serine-containing peptides, acetaldehyde (CH3CHO) from threonine-containing peptides, and 4-methylene-2,5-cycohexadienone (C7H6O) from tyrosine-containing peptides were generally observed in the negative ion PSD and SORI-CID spectra. Side-chain loss occurs much less from tyrosine-containing peptides than from serine- and threonine-containing peptides. This is probably due to the bulky side chain of tyrosine, resulting in steric hindrance and poor geometry for dissociation reactions. Additionally, a selective cleavage leading to the elimination of the C-terminal residue from [M,H], was observed from the peptides with serine and threonine at the C-terminus. This cleavage does not occur in the dissociation of peptides with an amide group at the C-terminus or peptides with neutral or basic residues at the C-terminus. It also does not occur with tyrosine at the C-terminus. Both the C-terminal carboxylic acid group and the hydroxyl side chain of the C-terminal residue must play important roles in the mechanism of C-terminal residue loss. A mechanism involving both the C-terminal carboxylic acid group and a hydroxyl side chain of serine and threonine is proposed. Copyright © 2007 John Wiley & Sons, Ltd. [source] Porous polymer monolith for surface-enhanced laser desorption/ionization time-of-flight mass spectrometry of small moleculesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2004Dominic S. Peterson Porous poly(butyl methacrylate- co -ethylene dimethacrylate), poly(benzyl methacrylate- co -ethylene dimethacrylate), and poly(styrene- co -divinylbenzene) monoliths have been prepared on the top of standard sample plates used for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the modified plates were used for laser desorption/ionization mass spectrometry (LDI-MS). The hydrophobic porous surface of these monoliths enables the transfer of sufficient energy to the analyte to induce desorption and ionization prior to TOFMS analysis. Both UV and thermally initiated polymerization using a mask or circular openings in a thin gasket have been used to define spot locations matching those of the MALDI plates. The desorption/ionization ability of the monolithic materials depends on the applied laser power, the solvent used for sample preparation, and the pore size of the monoliths. The monolithic matrices are very stable and can be used even after long storage times in a typical laboratory environment without observing any deterioration of their properties. The performance of the monolithic material is demonstrated with the mass analysis of several small molecules including drugs, explosives, and acid labile compounds. The macroporous spots also enable the archiving of samples. Copyright © 2004 John Wiley & Sons, Ltd. [source] Enhanced post-source decay and cross-ring fragmentation of oligosaccharides facilitated by conversion to amino derivativesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2004Jan Muzikar Post-source decay (PSD) fragmentation of chemically or enzymatically produced aminoglycans has been evaluated through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Conversion of native glycans to their respective aminoglycan derivatives improved detection sensitivity of the usual fragments and promoted cross-ring fragmentation of linear oligosaccharides, facilitating linkage recognition. The cross-ring fragmentations for both dextrin and dextran oligosaccharides were not limited to the reducing-end glucose moiety, as they were extended throughout the entire molecule. When the amino group was generated for N-glycans derived from three different glycoproteins, an enhancement of PSD was observed, without a significant extent of cross-ring fragmentation. Copyright © 2004 John Wiley & Sons, Ltd. [source] Characterization of an insoluble polyimide oligomer by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2002Anthony P. Gies In the past two years, papers have appeared in the literature which demonstrate that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra can be obtained from matrix-analyte preparations which have been produced by grinding the two materials together until a powder of small particle size is obtained. In the present study that methodology was modified and applied to an insoluble polyimide oligomer, poly(4,4,-oxydiphenylenepyromellitimide) (POPM). Two matrix materials were employed in this analysis, 1,8 dihydroxyanthrone (dithranol) and 3-aminoquinoline, with and without an additional cationizing agent. The spectra obtained by this method are shown to be sensitive to the matrix employed in the analysis as well as the quantity of cationizing agent combined with the matrix. Copyright © 2002 John Wiley & Sons, Ltd. [source] Use of matrix clusters and trypsin autolysis fragments as mass calibrants in matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2002William A. Harris Trypsin autolysis fragments and matrix clusters are often observed as intense peaks in mass spectra of protein digests. It is demonstrated that these can be exploited to improve the mass calibration of a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometer. Interpretation of some of the autolysis masses is complicated by the existence of disulfide bonds. Surprisingly large matrix clusters are often visible for ,-cyano-4-hydroxy-cinnamic acid. The fractional part of their masses differentiates them from protein digestion fragments. Copyright © 2002 John Wiley & Sons, Ltd. [source] Mass spectral studies on aryl-substituted N -carbamoyl/N -thiocarbamoyl narcotine and related compoundsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2002Shefali Aggarwal Positive ion mass spectral fragmentation of new N-carbamoyl/N-thiocarbamoyl derivatives of narcotine and compounds closely related to it are reported and discussed. The techniques used include electron impact (EI), fast-atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Prominent peaks in the mass spectra of these compounds appear to involve C-C bond cleavage , to the amine nitrogen with loss of the 4,5-dimethoxy(1H)isobenzofuranone moiety from their molecular ions, along with another prominent peak at m/z 382. No molecular ion peaks of these compounds were recorded in EI, whereas intense [M,+,H]+ ion peaks were observed in FAB and ESI spectra. MALDI also yielded [M,+,H]+ ion peaks in good agreement with FAB and ESI studies. Copyright © 2002 John Wiley & Sons, Ltd. [source] Characterization of a [(O3/2SiMe)x(OSi(OH)Me)y(OSiMe2)z] silsesquioxane copolymer resin by mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2001Ron E. Tecklenburg Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were applied to a complex silsesquioxane-siloxane copolymer resin. The wide-polydispersity starting material was fractionated into 21 separate fractions in order to facilitate the analysis by mass spectrometry. ESI-FTICR exact mass measurements were able to identify the specific oligomers present in the lowest mass fractions and showed that very few unreacted silanol groups remained, that is, topologically closed structures predominated. MALDI-TOFMS was able to show that gel-permeation chromatography substantially underestimated the molecular masses of the higher mass fractions. Mass autocorrelation was able to show that the silsesquioxane monomer appeared only in even numbers in any given oligomer. This is a natural consequence of the highly condensed nature of the resin. Copyright © 2001 John Wiley & Sons, Ltd. [source] Characterization of tetrathiofulvalene compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2001Shaoxiang Xiong Tetrathiofulvalene compounds are important components of charge-transfer complexes, which may be applied in various fields of scientific research and practical applications. Some of these compounds cannot be characterized by mass spectrometry. Here, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for the characterization of tetrathiofulvalenes. The samples could be easily desorbed and ionized to form singly charged ions, and mass spectra with isotopic resolution readily obtained. The mass spectrometric results for 26 compounds have shown that MALDI-TOF is more effective and convenient than other mass spectrometry methods, and resolves the problem of mass spectrometric characterization of tetrathiofulvalene compounds. Copyright © 2001 John Wiley & Sons, Ltd. [source] Peptide products of the afp-6 gene of the nematode Ascaris suum have different biological actionsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2007Joanne Y. Yew Abstract Matrix-assisted laser desorption/ionization time-of-flight and tandem time-of-flight (MALDI-TOF and MALDI-TOF/TOF) mass spectrometry were used to sequence and localize three novel, related neuropeptides in the nervous system of the nematode Ascaris suum, AMRNALVRFamide (AF21), NGAPQPFVRFamide (AF22), and SGMRNALVRFamide (AF23). The amino acid sequences were used to clone a novel neuropeptide gene (afp-6) that encodes a precursor bearing a single copy of each of the peptides. In situ hybridization and immunocytochemistry revealed that both the transcript and the peptides are expressed in a single cell in the ventral ganglion. Pharmacological studies of intact nematodes injected with these peptides, as well as physiological studies of responses to them in muscle tissue, motor neurons, and the pharynx, reveal that these peptides have potent bioactivity in the locomotory and feeding systems. Further exploration of their effects may contribute to our understanding of neuropeptide modulation of behavior and also to the development of compounds with anthelmintic relevance. J. Comp. Neurol. 502:872,882, 2007. © 2007 Wiley-Liss, Inc. [source] Differential Capture of Serum Proteins for Expression Profiling and Biomarker Discovery in Pre- and Posttreatment Head and Neck Cancer Samples,THE LARYNGOSCOPE, Issue 1 2008Gary L. Freed MD Abstract Introduction: A long-term goal of our group is to develop proteomic-based approaches to the detection and use of protein biomarkers for improvement in diagnosis, prognosis, and tailoring of treatment for head and neck squamous cell cancer (HNSCC). We have previously demonstrated that protein expression profiling of serum can identify multiple protein biomarker events that can serve as molecular fingerprints for the assessment of HNSCC disease state and prognosis. Methods: An automated Bruker Daltonics (Billerica, MA) ClinProt matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer was used. Magnetic chemical affinity beads were used to differentially capture serum proteins prior to MALDI-TOF analysis. The resulting spectra were analyzed using postprocessing software and a pattern recognition genetic algorithm (ClinProt 2.0). An HNSCC cohort of 48 sera samples from 24 patients consisting of matched pretreatment and 6 to 12 month posttreatment samples was used for further analysis. Low-mass differentially expressed peptides were identified using MALDI-TOF/TOF. Results: In the working mass range of 1,000 to 10,000 m/z, approximately 200 peaks were resolved for ionic bead capture approaches. For spectra generated from weak cation bead capture, a k-nearest neighbor genetic algorithm was able to correctly classify 94% normal from pretreatment HNSCC samples, 80% of pretreatment from posttreatment samples, and 87% of normal from posttreatment samples. These peptides were then analyzed by MALDI-TOF/TOF mass spectometry for sequence identification directly from serum processed with the same magnetic bead chemistry or alternatively after gel electrophoresis separation of the captured proteins. We were able to compare this with similar studies using surface-enhanced laser desorption ionization (SELDI)-TOF to show this method as a valid tool for this process with some improvement in the identification of our groups. Conclusions: This initial study using new high-resolution MALDI-TOF mass spectrometry coupled with bead fractionation is suitable for automated protein profiling and has the capability to simultaneously identify potential biomarker proteins for HNSCC. In addition, we were able to show improvement with the MALDI-TOF in identifying groups with HNSCC when compared with our prior data using SELDI-TOF. Using this MALDI-TOF technology as a discovery platform, we anticipate generating biomarker panels for use in more accurate prediction of prognosis and treatment efficacies for HNSCC. [source] Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivityBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2007Y. Nakano Summary Background, Nonspecific unresponsive states of delayed-type hypersensitivity (DTH) to unrelated antigens are induced in mice by a single administration of hapten. In these studies, we found a unique regulatory mechanism of contact hypersensitivity (CHS) mediated by nonspecific suppressor factor (NSF) induced by the intravenous injection of hapten-conjugated syngeneic spleen cells. NSF is a , 45-kDa protein released from the macrophage-like suppressor cells and binds selectively to dendritic cells (DCs). Moreover, NSF-treated DCs release a second , 20-kDa NSF (NSFint). Objectives, To try and identify NSF and characterize its function. Methods, The suppressor activity was evaluated by inhibition of the passive transfer of CHS by the effector cells sensitized with hapten and the antigen-presenting cell (APC) activity of hapten-primed draining lymph node cells (DLNCs) to induce CHS. NSF-containing supernatants obtained from the culture of spleen cells from mice that had been injected intravenously with oxazolone-conjugated syngeneic spleen cells 7 days before were prepared and purified with a Green A dye-affinity column, DEAE column and Sephacryl S-200 column. Then, samples of molecular mass of , 45 kDa were separated by native-PAGE (polyacrylamide gel electrophoresis) and nonreducing sodium dodecyl sulphate (SDS)-PAGE. After confirming the suppressor activity of proteins of , 45 kDa separated by native-PAGE, samples were separated by nonreducing SDS-PAGE, transferred onto polyvinylidene difluoride membranes and analysed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Results, Proteins of , 45 kDa eluted from a Sephacryl S-200 column and the slice of native-PAGE gel exhibited the strong suppressor activity. Analyses using MALDI-TOF mass spectrometry and MASCOT algorithm of the protein bands around 45 kDa separated by nonreducing SDS-PAGE identified NSF as a 22·5-kDa protein, dual specific phosphatase 14/MAP-kinase phophatase-6 (DUSP14/MKP6), which functions as a negative regulator of the MAP-kinase signalling. Western blot analyses revealed that recombinant DUSP14 (rDUSP14) exists as the mixture of 22·5-kDa monomer and 45-kDa dimer under nonreducing conditions, and monomers under reducing conditions. Treatment with rDUSP14 at 4 °C for 2 h suppressed the ability of effector cells to transfer CHS dose dependently and the APC function of DLNCs to induce CHS. Epicutaneous application of rDUSP14 immediately after challenge inhibited the subsequent CHS expression. rDUSP14 was bound specifically by major histocompatibility complex class II (Ia)-positive spleen cells (presumably DCs). The suppressor activity of NSF was neutralized by anti-DUSP14 monoclonal antibody. Expression of DUSP14 mRNA in the spleen was upregulated parallel to the unresponsive state induced by hapten-conjugated cells. NSF, NSFint and rDUSP14 exhibited the phosphatase activity towards p -nitrophenyl phosphate in vitro as alkaline phosphatase. Conclusions, These studies indicate for the first time that NSF is a dimer of DUSP14 secreted by macrophage-like suppressor cells by stimulation with hapten-conjugated cells and exerts a regulatory function on CHS through DCs as a secreted phosphatase. [source] Exact mass measurement on an electrospray ionization time-of-flight mass spectrometer: error distribution and selective averagingJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2003Jiejun Wu Abstract An automated, accurate and reliable way of acquiring and processing flow injection data for exact mass measurement using a bench-top electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Using Visual Basic programs, individual scans were selected objectively with restrictions on ion counts per second for both the compound of interest and the mass reference peaks. The selected ,good scans' were then subjected to two different data-processing schemes (,combine-then-center' and ,center-then-average'), and the results were compared at various ion count limit settings. It was found that, in general, the average of mass values from individual scans is more accurate than the centroid mass value of the combined (same) scans. In order to acquire a large number of good scans in one injection (to increase the sampling size for statistically valid averaging), an on-line dilution chamber was added to slow down the typically rapid mass chromatographic peak decay in flow-injection analysis. This simple addition worked well in automation without the need for manual sample dilution. In addition, by dissolving the reference compound directly into the mobile phase, manual syringe filling can be eliminated. Twenty-seven samples were analyzed with the new acquisition and process routines in positive electrospray ionization mode. For the best method found, the percentage of samples with RMS error less than 5 ppm was 100% with repetitive injection data (6 injections per sample), and 95% with single injection data. Afterwards, 31 other test samples were run (with MW ranging from 310 to 3493 Da, 21 samples in ESI+ and 10 in ESI, mode) and processed with similar parameters and 100% of them were mass-calculated to RMS error less than 5 ppm also. Copyright © 2003 John Wiley & Sons, Ltd. [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] Mass measurement accuracy comparisons between a double-focusing magnetic sector and a time-of-flight mass analyzerRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2008Michael S. Bereman We report a direct comparison of the mass measurement accuracies (MMAs) obtained on different mass spectrometry instrument types; a magnetic sector as the ,gold standard' and an electrospray ionization time-of-flight (ESI-TOF) instrument. Sixty samples, obtained from the Department of Chemistry at North Carolina State University, were analyzed on each instrument. Data are presented and compared between the different instruments. The average absolute MMAs achieved for the magnetic sector and Agilent ESI-TOF mass spectrometers were 3.0 and 1.1,ppm, respectively. Copyright © 2008 John Wiley & Sons, Ltd. [source] Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24BIOTECHNOLOGY PROGRESS, Issue 5 2001Sven E. Kemminer R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 ,g sialic acid per mg protein, which splits into 0.243 ,g Neu5Gc and 0.217 ,g Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy. [source] | ||||||||||||||||