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Electrospray Ionization Mass Spectrometry (electrospray + ionization_mass_spectrometry)
Kinds of Electrospray Ionization Mass Spectrometry Selected AbstractsApplication of Electrospray Ionization Mass Spectrometry to the Elucidation of the Primary Structure of LigninMACROMOLECULAR BIOSCIENCE, Issue 7 2003Dmitry V. Evtuguin Abstract Electrospray ionization mass spectrometry (ESI-MS) was successfully applied to the structural analysis of lignin. The structure of oligomers fractionated from Eucalyptus globulus dioxane lignin was elucidated using tandem mass spectrometry, and the information on fragmentation patterns was provided by experiments on dimeric model compounds. Data obtained revealed a significant abundance in the lignin macromolecules of linear fragments that were composed of 8- O -4,-linked syringyl/guaiacyl units and syringaresinol. The proposed linear fragment of the E. globulus lignin molecule. [source] Innentitelbild: Sensitive Detection of Native Proteins Using Extractive Electrospray Ionization Mass Spectrometry (Angew. Chem.ANGEWANDTE CHEMIE, Issue 17 201017/2010) Die Charakterisierung nativer Proteine gelingt durch extrahierende Elektrosprayionisations-Massenspektrometrie (EESI-MS). Wie H. Chen und Mitarbeiter in ihrer Zuschrift auf S.,3117,ff. ausführen, werden native Proteine bei der EESI mit Ladungen belegt, ohne dass ein starkes elektrisches Feld benötigt wird. Das Verfahren erleichtert massenspektrometrische Studien, auch an biologischen Rohproben, und vermeidet signifikante Konformationsänderungen und Aktivitätsverluste. [source] Sensitive Detection of Native Proteins Using Extractive Electrospray Ionization Mass Spectrometry,ANGEWANDTE CHEMIE, Issue 17 2010Huanwen Chen Prof. ,Sanfte" Ionisation: Bei der EESI werden Ladungen auf native Proteine übertragen, ohne sie einem starken elektrischen Feld auszusetzen. So lassen sich Proteine in nichtaufbereiteten biologischen Proben massenspektrometrisch charakterisieren, ohne dass bedeutende Konformationsänderungen oder Aktivitätsverluste eintreten. Die EESI-MS bietet sich für die Hochdurchsatzanalyse kleinster Proteinmengen unter nativen Bedingungen an. [source] Probing the Subunit-Subunit Interaction of the Tetramer of E. coli KDO8P Synthase by Electrospray Ionization Mass Spectrometry,CHINESE JOURNAL OF CHEMISTRY, Issue 1 2009Zhili LI Abstract Escherichia coli 3-Deoxy- D - manno- octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation reaction between D -arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi). This enzyme exists as a tetramer in solution, which is important for catalysis. Two different states of the enzyme were obtained: i) PEP-bound and ii) PEP-unbound. The effect of the substrates and products on the overall structure of KDO8P synthase in both PEP-bound and unbound states was examined using electrospray ionization mass spectrometry. The analysis of our data showed that the complexes of the PEP-unbound enzyme with PEP (or Pi) favored the formation of monomers, while the complexes with A5P (or KDO8P) mainly favored dimers. The PEP-bound enzyme was found to exist in the monomer and dimer with a small amount of the tetramer, whereas the PEP-unbound form primarily exists in the monomer and dimer, and no tetramer was observed, suggesting that the bound PEP have a role in stabilization of the tetrameric structure. Taken together, the results imply that the addition of the substrates or products to the unbound enzyme may alter the subunit-subunit interactions and/or conformational change of the protein at the active site, and this study also demonstrates that the electrospray ionization mass spectrometric method may be a powerful tool in probing the subunit-subunit interactions and/or conformational change of multi-subunit protein upon binding to ligand. [source] Organization of nucleobase-functionalized ,-peptides investigated by soft electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2009Nicola Diezemann Abstract The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI-MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) as well as a quadrupole/time-of-flight tandem mass spectrometer (QqToF-MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical ,-peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of ,-peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR-MS and QqToF-MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)-MS/MS and infrared multiphoton dissociation (IRMPD)-MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd. [source] Mass spectrometric analysis of 7-sulfoxymethyl-12-methylbenz[a]anthracene and related electrophilic polycyclic aromatic hydrocarbon metabolitesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2004Andreas F. Lehner Abstract The Meso-region theory of polycyclic aromatic hydrocarbon (PAH) carcinogenesis predicts that the development of pronounced carcinogenicity depends on the introduction of a good leaving group on alkyl side-chains attached to the exceptionally reactive meso-anthracenic or L-region positions of PAHs. Thus, the first step in carcinogenesis by methylated PAHs such as 7,12-dimethylbenz[a]anthracene (DMBA) would be the hydroxylation of the L-region methyl groups, particularly the 7-methyl group. The second would be the formation of a metabolite, e.g. a sulfate ester, which is expected to be a good leaving group capable of generating a highly reactive benzylic carbocation. 7-Hydroxymethyl-12-methylbenz[a]anthracene (7-HMBA) is a metabolite of DMBA, and sulfation of 7-HMBA to a 7-sulfoxymethyl metabolite (7-SMBA) is a known Phase II metabolic process designed to facilitate excretion, but actually enabling more destructive side-reactions. These side-reactions occur with generation of an electrophilic 7-methylene carbonium ion, and/or by in vivo halide exchange to provide neutral side-products more capable of entering cells, especially those of DMBA target tissues. Electrospray ionization mass spectrometry (MS) enabled us to visualize 7-SMBA as an intact m/z 351 conjugate anion by negative mode, and as a released m/z 255 carbonium ion by positive mode. Upon prolonged refrigeration, 7-SMBA accumulated an m/z 383 photooxide, which appeared capable of re-evolving the starting material as visualized by tandem quadrupole MS, or MS/MS. The 7-SMBA carbonium ion provided interpretable fragments when studied by fragment ion MS/MS, including those representing the loss of up to several protons. Subtle differences in this property were encountered upon perturbing 7-SMBA, either by warming it at 37 °C for 2 h or by substituting the initial sulfoxy group with an iodo group. Side-reactions accounting for such proton losses are proposed, and are of interest whether they occur in the mass spectrometer, in solution or both; these proposals include acidity at the 12-methyl position and cyclization between the 12-methyl group and the adjacent C-1 position. It is also suggested that such side-reactions may comprise one route to relieving steric strain arising between the 12-methyl group and the angular benzo ring of 7-SMBA. Copyright © 2004 John Wiley & Sons, Ltd. [source] Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2003Herbert Oberacher Abstract Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4, charge state of (dT)24 (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12,18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24, charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography. Copyright © 2003 John Wiley & Sons, Ltd. [source] Application of Electrospray Ionization Mass Spectrometry to the Elucidation of the Primary Structure of LigninMACROMOLECULAR BIOSCIENCE, Issue 7 2003Dmitry V. Evtuguin Abstract Electrospray ionization mass spectrometry (ESI-MS) was successfully applied to the structural analysis of lignin. The structure of oligomers fractionated from Eucalyptus globulus dioxane lignin was elucidated using tandem mass spectrometry, and the information on fragmentation patterns was provided by experiments on dimeric model compounds. Data obtained revealed a significant abundance in the lignin macromolecules of linear fragments that were composed of 8- O -4,-linked syringyl/guaiacyl units and syringaresinol. The proposed linear fragment of the E. globulus lignin molecule. [source] Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometryPROTEIN SCIENCE, Issue 2 2000Randy M. Whittal Abstract Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KD's are <100 nm. n-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both ph's. cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes. [source] The relationship between the electrospray ionization behaviour and biological activity of some phosphino Cu(I) complexesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2010Francesco Tisato Electrospray ionization mass spectrometry was usefully employed for the characterization of three phosphino copper(I) complexes of medicinal interest. This technique revealed that the original [CuL4]+ pro-drugs (L,=,hydrophilic tertiary phosphine) underwent dissociation with production of coordinative unsaturated [CuL3]+ and [CuL2]+ species, which represented key intermediates for the activation of potential biological properties. The more favoured was the displacement of the ligands from the [CuL4]+ parent complex, the more favoured was in turn the possibility for the metal ion to directly interact with biological substrates, including pharmacological targets related to cancer proliferation. An inverse correlation between the stability and the cytotoxic activity of the three copper(I) complexes investigated has been clearly established. Copyright © 2010 John Wiley & Sons, Ltd. [source] Perturbations produced by electrospray ionization mass spectrometry in the speciation of aluminium(III)/1,6-dimethyl-4-hydroxy-3-pyridinecarboxylate aqueous solutionsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2010Valerio B. Di Marco Electrospray ionization mass spectrometry (ESI-MS) is very often employed to study metal/ligand equilibria in aqueous solution. However, the ionization process can introduce perturbations which affect the speciation results in an unpredictable way. It is necessary to identify these perturbations in order to correctly interpret the ESI-MS speciation results. Aluminium(III)/1,6-dimethyl-4-hydroxy-3-pyridinecarboxylate (DQ716) aqueous solutions at various pH were analysed by ESI-MS, and speciation results were compared with those obtained by equilibrium techniques. Differences observed were both qualitative and quantitative. The ESI-MS spectral changes due to different settings of the following instrumental parameters were analyzed: the solution flow rate (FS), the nebulizer gas flow rate (FG), the potential applied at the entrance capillary (EC), and the temperature of the drying gas (TG). The effects produced by FS and EC on the spectra strongly suggest the key role of surface activity in determining the relative fraction of the ions reaching the detector. The experimental effects of FS and TG were interpreted considering the presence of at least two reactions in the gas phase and a dimerization occurring in the droplets. These perturbations cannot be generalized because they appear to be chemical system-related and instrument-dependent. Therefore, the identification of perturbations is a required task for any metal-ligand equilibrium study performed by ESI-MS. Our results indicate that perturbations can be identified by evaluating the effects produced in the spectra by a change of instrumental parameters. Copyright © 2010 John Wiley & Sons, Ltd. [source] Complexation of late transition metal(II) ions (M,=,Co, Ni, Cu, and Zn) by a macrocyclic thiacrown ether studied by means of electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2009Nikos G. Tsierkezos Electrospray ionization mass spectrometry (ESI-MS) is used to probe the metal-binding selectivity of a macrocyclic thiacrown ether (C44H32S20) towards CoII, NiII, CuII, and ZnII. In homogeneous 1:1 v/v methanol/dichloromethane solutions, it is found that the thia ligand very selectively binds traces of copper even in the presence of an excess of the other metal ions. The large selectivity is ascribed to the redox-active nature of copper which enables a reduction from CuII to CuI, occurring upon ESI-MS, whereas CoII, NiII and ZnII cannot undergo similar redox reactions. Copyright © 2009 John Wiley & Sons, Ltd. [source] Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2005Alexandru C. Lazar Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd. [source] Electrospray ionization mass spectrometry in studies of aluminium(III),ligand solution equilibriaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2003Valerio B. Di Marco Electrospray ionization mass spectrometry (ESI-MS) has been applied to the study of solution equilibria between Al(III) and the two ligands 4-hydroxy-3-pyridinecarboxylic acid (4H3P) and 3-hydroxy-4-pyridinecarboxylic acid (3H4P). The results compare well with the speciation data obtained from potentiometric, UV-visible spectroscopy, and NMR measurements. This agreement suggests the applicability of ES-MS to the study of more complicated aluminium,ligand systems. Copyright © 2003 John Wiley & Sons, Ltd. [source] Electrospray ionization mass spectrometry of AZT H -phosphonates conjugated with steroidsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2003Qiang Xiao AZT H -phosphonates conjugated with steroids were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated in detail. There are very different characteristic fragment ions in the positive and negative ion MS/MS spectra. The azide group of compounds 6a and 6b underwent either elimination of HN3 or rearrangement to an amine in both positive and negative ion mass spectrometry. Copyright © 2003 John Wiley & Sons, Ltd. [source] Electrospray ionization mass spectrometry of serine/alanine conjugated 5,-UMP and 3,,5,-dithymidine phosphoramidatesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2002Zhi-Hui Qin First page of article [source] Hybridization of short complementary PNAs to G-quadruplex forming oligonucleotides: An electrospray mass spectrometry studyBIOPOLYMERS, Issue 4 2009Jussara Amato Abstract We investigated the interaction of the short peptide nucleic acid (PNA) strand [acccca]-PNA with oligodeoxynucleotides containing one, two, or four tracts of TGGGGT units. Electrospray ionization mass spectrometry allowed exploring the wide variety of complex stoichiometries that were found to coexist in solution. In water, the PNA strand forms short heteroduplexes with the complementary DNA sequences, but higher-order structures are also found, with PNA2n·DNAn triplex units, culminating in precipitation at very low ionic strength. In the presence of ammonium acetate, there is a competition between PNA·DNA heteroduplex formation and DNA G-quadruplex formation. Heteroduplex formation is favored when the PNA + DNA mixture in ammonium acetate is heated and cooled at room temperature, but not if the PNA is added at room temperature to the preformed G-quadruplex. We also found that the short [acccca]-PNA strand binds to G-quadruplexes. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 244,255, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Identification and quantification of in vitro adduct formation between protein reactive xenobiotics and a lysine-containing model peptideENVIRONMENTAL TOXICOLOGY, Issue 1 2003Peter Reichardt Abstract Formation of in vitro adducts between different classes of xenobiotics and the lysine-containing peptide Lys-Tyr was monitored by high-performance liquid chromatography and electrospray ionization mass spectrometry. The molecular structures of the main resulting products could be sensitively analyzed by mass spectrometry (flow injection analysis), enabling the detection of characteristic binding formations. Aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde were shown to form stable linkages to lysine amino groups via Schiff bases. Other electrophilic substances (e.g., toluene-2,4-diisocyanate, 2,4-dinitro-1-fluorobenzene, 2,4,6-trinitrobenzene sulfonic acid, dansyl chloride, and phthalic acid anhydride) also formed covalent adducts with lysine residues. The reactivity of the compounds was quantified by measuring the amount of peptide that remained unchanged after incubation for a certain period with the xenobiotic. Although reactivity levels within this group of aldehydes varied only to a small extent, as would be expected, extreme differences were seen among the structurally heterogeneous group of nonaldehyde xenobiotics. These results support the hypothesis that simple chemical reactions may lead to the adduction of nucleophilic macromolecules such as peptides or proteins. Such reactions, in particular, Schiff base formation of aldehydes, have previously been shown to be capable of specifically interfering with costimulatory signaling on T cells. Our results suggest that electrophilic xenobiotics of other classes may also inherit the capacity to exert similar effects. Forming covalent linkage to peptides may represent a possible molecular mechanism of electrophilic xenobiotics in vivo, yielding immunotoxic effects. The model utilized in this study is appropriate for monitoring the adduction of xenobiotics to basic peptides and for analyzing the resulting molecular structures. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 29,36, 2003. [source] Determination and fate of oxytetracycline and related compounds in oxytetracycline production wastewater and the receiving river,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2008Dong Li Abstract This study investigated the occurrence and fate of oxytetracycline (OTC) and its related substances, 4-epi-oxytetracycline (EOTC), ,-apo-oxytetracycline (,-apo-OTC), and ,-apo-oxytetracycline (,-apo-OTC), in a wastewater treatment plant (WWTP) treating OTC production wastewater and a river receiving the effluent from the WWTP using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The percent removal of OTC in the WWTP was 38.0 ± 10.5%, and the concentration of OTC was still up to 19.5 ± 2.9 mg/L in the treated outflow. The concentration slightly decreased along the river, from 641 ± 118 ,g/L at site R2 (discharging point) to 377 ± 142 ,g/L at site R4 (,20 km from site R2), which was still higher than the minimal inhibition concentration of OTC reported (,250 ,g/L). On the other hand, the total amount of its related substances in the treated effluent was less than 5% of OTC. Concentrations of ,-apo-OTC and ,-apo-OTC increased along the river, from 5.76 ± 0.63 and 2.08 ± 0.30 ,g/L at site R2 to 11.9 ± 4.9 and 12.0 ± 4.6 ,g/L at R4, respectively, although EOTC decreased from 31.5 ± 3.8 to 12.9 ± 1.1 ,g/L, respectively. The mean concentration of ,-apo-OTC in river sediments was 20.8 ± 7.8 mg/kg, and its ratio to OTC was approximately 0.11, nearly twice the ratio of ,-apo-OTC and EOTC to OTC (0.058 ± 0.014 and 0.061 ± 0.015, respectively). [source] Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogenFEBS JOURNAL, Issue 2 2008Outi Itkonen Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein,protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens. [source] Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magnaFEBS JOURNAL, Issue 14 2006Tobias Lamkemeyer The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 ± 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 ± 11.1 kDa (hemoglobin-rich animals) and 597.5 ± 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 ± 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm. [source] Haemoglobin Pierre,Benite , a high affinity variant associated with relative polycythaemiaINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 6 2001M.E.J. Beard This is the second reported example of Hb Pierre,Benite (,90 Glu,Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal , chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants , Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia. [source] CHRACTERIZATION AND 1,1-DIPHENYL-2-PICRYLHYDRAZYL RADICAL SCAVENGING ACTIVITY OF METHANOL AND SUPERCRITICAL CARBON DIOXIDE EXTRACTS FROM LEAVES OF ADINANDRA NITIDAJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2008BENGUO LIU ABSTRACT Leaves of Adinandra nitida are consumed in southern China as health tea (Shiyacha) and as herbal medicine. In this study, the methanol and supercritical fluid extracts from leaves of A. nitida were obtained by traditional solvent extraction and supercritical carbon dioxide extraction, respectively. Both the extracts showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. By using ultraviolet-visible spectrometry (UV), infrared spectrometry (IR), nuclear magnetic resonance, electrospray ionization mass spectrometry (ESI-MS), high-performance liquid chromatography-ESI/MS, the main bioactive constituents in the methanol extract (ME) were identified as camellianin A, camellianin B, apigenin. By analysis of gas chromatography-mass spectrometry, a total of 16 compounds accounting for 98.79% of the supercritical fluid extract (SFE) were identified as ,-sitosterol, vitamin E, ,-tocopherol and so on. These compounds found in ME and SFE could contribute to the DPPH radical scavenging performance of the extracts in this study. PRACTICAL APPLICATION Adinandra nitida is a kind of particular wild plant in South China. Few reports have been published about it in the world. In this study, the methanol and supercritical fluid extracts from leaves of A. nitida were respectively obtained by two kinds of industrially significant methods, traditional solvent extraction and supercritical carbon dioxide extraction. By using ultraviolet-visible spectrometry (UV), infrared spectrometry (IR), nuclear magnetic resonance, electrospray ionization mass spectrometry (ESI-MS), high-performance liquid chromatography-ESI/MS, gas chromatography-MS, the main bioactive constituents in the two extracts were identified as flavonoids and plant sterols. Both the extracts showed high 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and this activity of the flavonoid-rich methanol extract was 10 times more than that of butylated hydroxytoluene. These results showed that leaves of A. nitida is a new kind of natural antioxidant-rich, flavonoid-rich plant source with great commercial interest in the food and phytopharmaceutical market. [source] IDENTIFICATION OF PROCYANIDIN A2 AS POLYPHENOL OXIDASE SUBSTRATE IN PERICARP TISSUES OF LITCHI FRUITJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2007JIAN SUN ABSTRACT Postharvest browning of litchi fruit results in short shelf life and reduced commercial value. Experiments were conducted to separate, purify and identify polyphenol oxidase (PPO ) substrates that cause litchi fruit to brown. PPO and its substrate were extracted from the pericarp tissues of litchi fruit. The litchi PPO substrate was purified using polyamide column, silica gel column and Sephadex LH-20 column chromatography. The browning substrate was selected by a 0.5% FeCl3 solution and then identified using a partially purified litchi PPO. Analyses of ultraviolet spectrometry, nuclear magnetic resonance and electrospray ionization mass spectrometry indicated that the PPO substrate was procyanidin A2. The substrate can be oxidized to , -quinones by litchi PPO and then form brown-colored by-products, resulting in pericarp browning of harvested litchi fruit. [source] Tandem mass spectrometry of synthetic polymersJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2009Anna C. Crecelius Abstract The detailed characterization of macromolecules plays an important role for synthetic chemists to define and specify the structure and properties of the successfully synthesized polymers. The search for new characterization techniques for polymers is essential for the continuation of the development of improved synthesis methods. The application of tandem mass spectrometry for the detailed characterization of synthetic polymers using the soft ionization techniques matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS), which became the basic tools in proteomics, has greatly been increased in recent years and is summarized in this perspective. Examples of a variety of homopolymers, such as poly(methyl methacrylate), poly(ethylene glycol), as well as copolymers, e.g. copolyesters, are given. The advanced mass spectrometric techniques described in this review will presumably become one of the basic tools in polymer chemistry in the near future. Copyright © 2009 John Wiley & Sons, Ltd. [source] Organization of nucleobase-functionalized ,-peptides investigated by soft electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2009Nicola Diezemann Abstract The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI-MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) as well as a quadrupole/time-of-flight tandem mass spectrometer (QqToF-MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical ,-peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of ,-peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR-MS and QqToF-MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)-MS/MS and infrared multiphoton dissociation (IRMPD)-MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd. [source] HPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2008Sofie P. Pasilis Abstract Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low Rf values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation. Published in 2008 by John Wiley & Sons, Ltd. [source] Analytical characterization of microfabricated SU-8 emitters for electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2008Tiina Sikanen Abstract We present a detailed optimization and characterization of the analytical performance of SU-8-based emitters for electrospray ionization mass spectrometry (ESI/MS). The improved SU-8 fabrication process presented here enhances patterning accuracy and reduces the time and cost of fabrication. All emitters are freestanding and enable sample delivery by both pressure-driven and spontaneous flows. The optimized emitter design incorporates a sharp, double-cantilevered tip implemented to the outlet of an SU-8 microchannel and provides highly sensitive ESI/MS detection. Moreover, the optimized design allows the use of relatively large microchannel dimensions (up to 200 × 50 µm2, w × h) without sacrificing the detection sensitivity. This is advantageous with a view of preventing emitter clogging and enabling reproducible analysis. The measured limits of detection for the optimized emitter design were 1 nM for verapamil and 4 nM for Glu-fibrinopeptide B with good quantitative linearities between 1 nM and 10 µM (R2 = 0.9998) for verapamil and between 4 nM and 3 µM (R2 = 0.9992) for Glu-fibrinopeptide B. The measured tip-to-tip repeatability for signal intensity was 14% relative standard deviation (RSD) (n = 3; 5 µM verapamil) and run-to-run repeatability 4,11% RSD (n = 4; 5 µM verapamil) for all individual emitters tested. In addition, long-term stability of < 2% RSD was maintained for timescales of 30 min even under free flow conditions. SU-8 polymer was also shown to be chemically stable against most of the tested electrospray solvents. Copyright © 2008 John Wiley & Sons, Ltd. [source] Evaluation of relative DNA binding affinities of anthrapyrazoles by electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2007Suncerae I. Smith Abstract Binding interactions of a new series of anthrapyrazoles (APs) with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/anthrapyrazole mixtures, and they show a correlation to the shift in melting point of the DNA measured from a previous study. Minimal sequence specificity was observed for the series of anthrapyrazoles. Upon collisionally activated dissociation of the duplex/anthrapyrazole complexes, typically ejection of the ligand was the dominant pathway for most of the complexes. However, for complexes containing AP2 or mitoxantrone, strand separation with the ligand remaining on one of the single strands was observed, indicative of a different binding mode or stronger binding. Copyright © 2007 John Wiley & Sons, Ltd. [source] Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy- trans -2-nonenalJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2006Giancarlo Aldini Abstract Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1 : 0.25 to 1 : 5 HSA : HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans. Copyright © 2006 John Wiley & Sons, Ltd. [source] | |||||||||||||||||||||||||||||||||||||