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Mass Spectrometric Analysis (mass + spectrometric_analysis)
Kinds of Mass Spectrometric Analysis Selected AbstractsSynthesis and Mass Spectrometric Analysis of CyclostellettaminesH, I, K and LEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 5 2006Achim Grube Abstract Very recently the new cyclostellettamines H, I, K and L were identified from a Brazilian sponge of the genus Pachychalina. They were isolated together with the known cyclostellettamines A,G in a mixture of only 1.5 mg. To obtain further material for biological investigations, the synthesis of the four new cyclostellettamines has been carried out. Since mass spectrometry plays an essential role in identifying these compounds a systematic analysis of the cyclostellettamines is discussed. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Modulation of gene expression by extracellular pH variations in human fibroblasts: A transcriptomic and proteomic studyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2003Maja A. Bumke Abstract Homeostasis of the intracellular ionic concentration, in particular that of hydrogen ions, is pivotal to the maintenance of cell function and viability. Nonetheless, pH fluctuations in both the intracellular and the extracellular compartments can occurr during development, in physiological processes and in disease. The influence of pH variations on gene expression has been studied in different model systems, but only for a limited number of genes. We have performed a broad range analysis of the patterns of gene expression in normal human dermal fibroblasts at two different pH values (in the presence and in the absence of serum), with the aim of getting a deeper insight into the regulation of the transcriptional program as a response to a pH change. Using the Affymetrix gene chip system, we found that the expression of 2068 genes (out of 12,565) was modulated by more than two-fold at 24, 48 or 72 h after the shift of the culture medium pH to a more acidic value, stanniocalcin 1 being a remarkable example of a strongly up-regulated gene. Genes displaying a modulated pattern of expression included, among others, cell cycle regulators (consistent with the observation that acidic pH abolishes the growth of fibroblasts in culture) and relevant extracellular matrix (ECM) components. Extracellular matrix protein 2, a protein with a restricted pattern of expression in adult human tissues, was found to be remarkably overexpressed as a consequence of serum starvation. Since ECM components, whose expression is controlled by pH, have been used as targets for biomolecular intervention, we have complemented the Affymetrix analysis with a two-dimensional polyacrylamide gel electrophoresis analysis of proteins which are differentially secreted by fibroblasts at acidic or basic pH. Mass spectrometric analysis of more than 650 protein spots allowed the identification of 170 protein isoforms or fragments, belonging to 40 different proteins. Some proteins were only expressed at basic pH (including, for instance, tetranectin), while others (e.g., agrin) were only detectable at acidic pH. Some of the identified proteins may represent promising candidate targets for biomedical applications, e.g., for antibody-mediated vascular targeting strategies. [source] Total Synthesis, Characterization, and Conformational Analysis of the Naturally Occurring Hexadecapeptide Integramide,A and a DiastereomerCHEMISTRY - A EUROPEAN JOURNAL, Issue 1 2010Marta De, Zotti Dr. Abstract Integramide,A is a 16-amino acid peptide inhibitor of the enzyme HIV-1 integrase. We have recently reported that the absolute stereochemistries of the dipeptide sequence near the C terminus are L -Iva14 - D -Iva15. Herein, we describe the syntheses of the natural compound and its D -Iva14 - L -Iva15 diastereomer, and the results of their chromatographic/mass spectrometric analyses. We present the conformational analysis of the two compounds and some of their synthetic intermediates of different main-chain length in the crystal state (by X-ray diffraction) and in solvents of different polarities (using circular dichroism, FTIR absorption, and 2D NMR techniques). These data shed light on the mechanism of inhibition of HIV-1 integrase, which is an important target for anti-HIV therapy. [source] Mass spectrometry for cultural heritage knowledge: gas chromatographic/mass spectrometric analysis of organic remains in Neolithic potsherds,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2001Pasquale Agozzino [source] ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activityFEBS JOURNAL, Issue 8 2002Nadia J. Kershaw The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ,,15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to >,95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate , and , subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an ,2,2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the , subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the , subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major , subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N -acetylornithine to glutamate, but not the formation of N -acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N -acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the , subunit. [source] Identification of a new functional target of haloperidol metabolite: implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acidJOURNAL OF NEUROCHEMISTRY, Issue 2 2006Hyeon Soo Kim Abstract Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3-(4-fluorobenzoyl) propionic acid (FBPA) and 4-(4-chlorophenyl)-4-piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine-induced extracellular signal-regulated kinase 1/2 phosphorylation, and it specifically affected mitogen-activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol-treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor-independent role of haloperidol. [source] Synthesis, characterization, and nucleotidic chain cleavage ability of uncharged water soluble poly(ethylene glycol)-fullerene derivatives with an amphiphilic characterJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 6 2008Daniele Vitalini Abstract Water-soluble fullerenes containing two poly(ethylene glycol) branches [Full-(PEG)2] were prepared starting from commercial poly(ethylene glycol)-monomethyl ethers and C60 [Full-(PEG)2]s chemical characterization was made by FT-IR, NMR, and MALDI-TOF mass spectrometric analyses. Their thermal stability was determined by TGA experiments. The capability of C60 -derivatives to induce oligonucleotide cleavage under visible light irradiation was also ascertained. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2154,2153, 2008 [source] Quantification of Alzheimer pathology in ageing and dementia: age-related accumulation of amyloid-,(42) peptide in vascular dementiaNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2006H. Lewis Clinicopathological observations suggest there is considerable overlap between vascular dementia (VaD) and Alzheimer's disease (AD). We used immunochemical methods to compare quantities of amyloid-, (A,) peptides in post mortem brain samples from VaD, AD subjects and nondemented ageing controls. Total A, peptides extracted from temporal and frontal cortices were quantified using a previously characterized sensitive homogenous time-resolved fluorescence (HTRF) assay. The HTRF assays and immunocapture mass spectrometric analyses revealed that the A,(42) species were by far the predominant form of extractable peptide compared with A,(40) peptide in VaD brains. The strong signal intensity for the peak representing A,(4,42) peptide confirmed that these N-terminally truncated species are relatively abundant. Absolute quantification by HTRF assay showed that the mean amount of total A,(42) recovered from VaD samples was approximately 50% of that in AD, and twice that in the age-matched controls. Linear correlation analysis further revealed an increased accumulation with age of both A, peptides in brains of VaD subjects and controls. Interestingly, VaD patients surviving beyond 80 years of age exhibited comparable A,(42) concentrations with those in AD in the temporal cortex. Our findings suggest that brain A, accumulates increasingly with age in VaD subjects more so than in elderly without cerebrovascular disease and support the notion that they acquire Alzheimer-like pathology in older age. [source] Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactionsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2001Shuqing Sun Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-,2a and anti-interferon-,2a monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible). Copyright © 2001 John Wiley & Sons, Ltd. [source] High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometryELECTROPHORESIS, Issue 6 2005Ya Jin Abstract A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1,M NaOH at 25°C. The recovery of this one-step extraction method was 34,73% for proteins <67,kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within ±0.01,0.10% deviation for all the proteins <36,kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34,ng for cytochrome,c, ,-lactalbumin, myoglobin, ,-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4,29.0,kDa), 340,ng for glyceraldehyde-3-phosphate dehydrogenase (35.6,kDa) and albumin (66.3,kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS. [source] First report of saxitoxin in Finnish lakes and possible associated effects on human healthENVIRONMENTAL TOXICOLOGY, Issue 3 2005Jarkko Rapala Abstract This study is the first report of saxitoxin in cyanobacterial blooms in Finland. Bloom samples (n = 50) were collected from Finnish freshwater sites during summer months of 2002 and 2003. These samples were screened for the presence of paralytic shellfish toxins (PSTs) using the Jellett rapid PSP screening test. Samples testing positive for PSTs (n = 7) were further analyzed with saxiphilin- and voltage-gated sodium channel [3H]-STX,binding radioreceptor assays and liquid chromatography using fluorescence and mass spectrometric analysis. The results indicated that saxitoxin (STX) was the only PST analogue in the samples and that it was present in high concentrations, as much as 1 mg L,1. Microscopic analysis revealed that 95%,100% of the phytoplankton in the positive samples consisted of Anabaena lemmermannii. The trophic status of lakes in which STX-containing blooms were found varied from oligotrophic to hypertrophic. All the lakes had high nitrogen-to-phosphorus ratios. In some instances, samples had been collected from sites where swimmers had reported adverse health effects, and in three such cases, reported adverse health effects were associated with sites from which samples testing positive for STX had been received. Symptoms of fever, eye irritation, abdominal pains, and skin rash were reported in children aged 2,10 years after exposure to the water. These were not the adverse human symptoms typical of STX poisoning; rather, they represented acute effects often reported following recreational exposure to cyanobacterial blooms. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 331,340, 2005 [source] Soil metaproteomics: a review of an emerging environmental science.EUROPEAN JOURNAL OF SOIL SCIENCE, Issue 6 2009Significance, methodology, perspectives Summary Soil is a dynamic system in which microorganisms perform important tasks in organic matter transformations and nutrient cycles. Recently, some studies have started to focus on soil metaproteomics as a tool for understanding the function and the role of members of the microbial community. The aim of our work was to provide a review of soil proteomics by looking at the methodologies used in order to illustrate the challenges and gaps in this field, and to provide a broad perspective about the use and meaning of soil metaproteomics. The development of soil metaproteomics is influenced strongly by the extraction methods. Several methods are available but only a few provide an identification of soil proteins, while others extract proteins and are able to separate them by electrophoresis but do not provide an identification. The extraction of humic compounds together with proteins interferes with the latter's separation and identification, although some methods can avoid these chemical interferences. Nevertheless, the major problems regarding protein identification reside in the fact that soil is a poor source of proteins and that there is not enough sequence-database information for the identification of proteins by mass spectrometric analysis. Once these pitfalls have been solved, the identification of soil proteins may provide information about the biogeochemical potential of soils and pollutant degradation and act as an indicator of soil quality, identifying which proteins and microorganisms are affected by a degradation process. The development of soil metaproteomics opens the way to proteomic studies in other complex substrates, such as organic wastes. These studies can be a source of knowledge about the possibility of driven soil restoration in polluted and degraded areas with low organic matter content and even for the identification of enzymes and proteins with a potential biotechnological value. [source] Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24FEBS JOURNAL, Issue 20 2000Toshiyuki Sakaki Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1,,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1,,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43,48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3 -26,23-lactol to 25(OH)D3 -26,23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1,,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1,,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. [source] Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjectsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2008Tsutomu Fujimura Abstract We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewisx/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the ,-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the ,-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAc,4(NeuAc,3)Gal,3(NeuAc,6)GlcNAc,Gal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O -glycosylation were identified in prostate cancer haptoglobin for the first time. Mono- and disialyl core Type 1 O-linked structures were identified after reductive ,-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O -glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N -glycans attached to a defined peptide region of its ,-chain are characterized by enhanced branching as well as antenna fucosylation. © 2007 Wiley-Liss, Inc. [source] Determination of primary bond scissions by mass spectrometric analysis of ultrasonic degradation products of poly(ethylene oxide- block -propylene oxide) copolymersJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2010Takehiro Watanabe Abstract Ultrasonic degradation of poly(ethylene oxide- block -propylene oxide) copolymers consisting of a hydrophilic and a hydrophobic portion was studied with the aim to determine the location of bonds involved in the initial scission of the copolymers. LC,APCI-IT-MS and LC,APCI-orbitrap-MS were used for the detailed structural analysis of degradation products. The results indicated that initial bond scissions occurred principally at the boundary regions between backbones of polyethylene oxide (PEO) and polypropylene oxide (PPO) chains. Further structural analysis revealed the presence of oxygen adducts in the degradation products. Comparison with a thermal degradation carried out in helium atmosphere, one can conclude that the oxygen adducts are formed by radical reaction with water or dissolving oxygen molecules. The study demonstrated that chemical reactions as well as physical bond stress scissions are involved in the ultrasonic degradation of the copolymers. Copyright © 2010 John Wiley & Sons, Ltd. [source] MALDI-TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varietiesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2009alplachta Abstract A procedure for identification of malting barley varieties using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ethanol-soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI-TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry-based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd. [source] Comparison of bovine and porcine ,-lactoglobulin: a mass spectrometric analysisJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2006Gaetano Invernizzi Abstract Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is applied to comparison of bovine and porcine ,-lactoglobulin (BLG and PLG). The conformational and oligomeric properties of the two proteins under different solvent and experimental conditions are analyzed. The pH-dependence of dimerization is described for the pH range 2,11. The results indicate maximal dimer accumulation at pH 6 for BLG and pH 4 for PLG, as well as a lower stability of the PLG dimer at pH 4 compared to BLG at pH 6. Conformational stability appears to be higher for BLG at acidic pH, but higher for PLG at basic pH. The higher stability of BLG at low pH is revealed by means of either chemical or thermal denaturation. Equilibrium folding intermediates of both proteins are detected. Finally, conditions are found that promote dissociation of the BLG dimer at pH 6 into folded monomers. Copyright © 2006 John Wiley & Sons, Ltd. [source] MALDI-TOF mass spectrometric analysis for the characterization of the 5,10,15,20-tetrakis- (m -hydroxyphenyl)bacteriochlorin (m -THPBC) photoproducts in biological environmentJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005Henri-Pierre Lassalle Abstract Photoproducts formation upon irradiation (739 nm) of 5,10,15,20-tetrakis(m -hydroxyphenyl)bacteriochlorin (m -THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption spectroscopy and MALDI-TOF mass spectrometry. The experiments were performed with a freshly prepared PBS,HSA solution of m -THPBC and with a PBS,HSA m -THPBC solution incubated for 6 h at 37 °C. The incubation of m -THPBC solution leads to the dye monomerisation, whereas in the freshly prepared solution, m -THPBC is under an aggregated form. Regardless of the incubation condition, photobleaching experiments carried out by absorption spectroscopy demonstrate the degradation of the photosensitizer and its phototransformation in m -THPC. Moreover, m -THPC was the sole photoproduct detected using absorption spectroscopy. Together with a degradation of m -THPBC and formation of m -THPC, MALDI-TOF mass spectrometry evidenced several other photoinduced modifications. Photoproducts such as dihydroxy m -THPBC and dihydroxy m -THPC were detected in both conditions; however, the formation of hydroxylated photoproducts was significantly greater in incubated solution. In addition, small molecules arising from the degradation of the photosensitizer and identified as dipyrin derivatives and dipyrrolic synthon were observed. Copyright © 2005 John Wiley & Sons, Ltd. [source] Glycan side chains on naturally presented MHC class II ligandsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2005Jörn Dengjel Abstract The molecular characterization of unknown naturally presented major histocompatibility complex (MHC) class II glycopeptides carrying complex glycans has so far not been achieved, reflecting the different fragmentation characteristics of sugars and peptides in mass spectrometric analysis. Human leukocyte antigen (HLA)-DR-bound peptides were isolated by affinity purification, separated via high performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. We were able to identify two naturally processed MHC class II ligands, CD53122,136 and CD53121,136, carrying complex N -linked glycan side chains by a combination of in-source and collision-induced fragmentation on a quadrupole time-of-flight tandem mass spectrometer. Copyright © 2005 John Wiley & Sons, Ltd. [source] A novel class of chemically modified iodo-containing resins: design, synthesis and application to mass spectrometry-based proteome analysisJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2004Li Zhang Abstract A novel class of chemically modified iodo-containing resins with isotope-labeled tagging for mass spectrometry-based proteome analysis is described. This iodo-containing resin contains a thiol-reactive group that is used to capture the cysteine (Cys)-containing peptides from peptide mixtures, one ,tag' amino acid, and an aminomethyl polystyrene resin with Rink Amide Linker. The ,tag' amino acid is synthesized in both heavy and light isotope-coded forms and therefore permits the direct relative quantification of peptides/proteins through mass spectrometric analysis. In the iodo-containing resin strategy, the Cys-containing peptides of two samples covalently captured by either light or heavy iodo-containing resin were mixed and washed extensively under stringent conditions. Then the Cys-containing peptides were retrieved by acid-catalyzed elution. Finally, the eluted peptides were directly analyzed by micro liquid chromatography/mass spectrometry for identification and relative quantification. The iodo-containing resins were synthesized by a simple but effective method. Their abilities to identify and quantify the Cys-containing part in two samples were proved by the analysis of mixtures of amino acids, peptides and proteins. Copyright © 2004 John Wiley & Sons, Ltd. [source] Influence of mobile phase composition on the high-performance liquid chromatographic/electrospray ionization mass spectrometric analysis of 11-nor-9-carboxy-,9 -tetrahydrocannabinol (THC-COOH) and its glucuronide in urineJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2004[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] Matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of oxygenated triglycerides and phosphatidylcholines in egg tempera paint dosimeters used for environmental monitoring of museum display conditionsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2001Oscar F. van den Brink Abstract Oxidative changes in triacylglycerols and diacylphosphatidylcholines in egg tempera paint strips are used for chemical dosimetry of the quality of the museum environment. High-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used as a rapid method for the determination of the exact elemental composition of the alteration products from diacylphosphatidylcholines and triacylglycerols. Light exposure of the egg tempera paints yields oxygenated diacylphosphatidylcholines and triacylglycerols. In the latter multiple incorporation of oxygen was observed as a recurring mass difference of 15.995, the exact atomic mass of oxygen. Owing to the high resolution of the FTMS data (routinely 20 000 at m/z 1000 in broadband mode), oxidation products with different elemental compositions but identical nominal mass could be distinguished. Products of oxidative cleavage of triacylglycerols were observed in samples exposed for longer times. The relative intensities of the peaks of singly and multiply oxygenated triacylglycerols were used to derive the degree of oxygenation of the egg lipids in the tempera paint dosimeters. The degree of oxygenation was found to be directly related to the light exposure time. Exposure to elevated temperature (60 °C) for a period of 21 days did not lead to oxygenation of the triacylglycerols and diacylphosphatidylcholines. Exposure to NOx and SO2 in the dark greatly increased the degree of oxygenation. Addition of lead- or copper-containing pigments to the egg binding medium (and subsequent storage for 6 months in the dark) led to accelerated conversion of egg lipids to oxidised products. Copyright © 2001 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] Chronic Ethanol Consumption Induces Global Hepatic Protein HyperacetylationALCOHOLISM, Issue 2 2010Blythe D. Shepard Background:, Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly. Methods:, To identify other hyperacetylated proteins in ethanol-fed livers, we chose a proteomics approach. Cytosolic and membrane proteins (excluding nuclei) were separated on 2D gels, transferred to PVDF and immunoblotted with antibodies specific for acetylated lysine residues. Hyperacetylated proteins were selected for trypsin digestion and mass spectrometric analysis. Results:, In all, 40 proteins were identified, 11 of which are known acetylated proteins. Remarkably, the vast majority of hyperacetylated membrane proteins were mitochondrial residents. Hyperacetylated cytosolic proteins ranged in function from metabolism to cytoskeletal support. Notably, 3 key anti-oxidant proteins were identified whose activities are impaired in ethanol-treated cells. We confirmed that the anti-oxidant enzyme, glutathione peroxidase 1, actin and cortactin are hyperacetylated in ethanol-treated livers. Conclusions:, Alcohol-induced hyperacetylation of multiple proteins may contribute to the development of liver injury. The abundance of acetylated mitochondrial proteins further suggests that this modification is important in regulating liver metabolism and when perturbed, may contribute to the progression of a variety of metabolic diseases. [source] Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2006Johan Jacksén Abstract A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated. [source] Shotgun lipidomics: Electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samplesMASS SPECTROMETRY REVIEWS, Issue 3 2005Xianlin Han Abstract Lipidomics, after genomics and proteomics, is a newly and rapidly expanding research field that studies cellular lipidomes and the organizational hierarchy of lipid and protein constituents mediating life processes. Lipidomics is greatly facilitated by recent advances in, and novel applications of, electrospray ionization mass spectrometry (ESI/MS). In this review, we will focus on the advances in ESI/MS, which have facilitated the development of shotgun lipidomics and the utility of intrasource separation as an enabling strategy for utilization of 2D mass spectrometry in shotgun lipidomics of biological samples. The principles and experimental details of the intrasource separation approach will be extensively discussed. Other ESI/MS approaches towards the quantitative analyses of global cellular lipidomes directly from crude lipid extracts of biological samples will also be reviewed and compared. Multiple examples of lipidomic analyses from crude lipid extracts employing these approaches will be given to show the power of ESI/MS techniques in lipidomics. Currently, modern society is plagued by the sequelae of lipid-related diseases. It is our hope that the integration of these advances in multiple disciplines will catalyze the development of lipidomics, and such development will lead to improvements in diagnostics and therapeutics, which will ultimately result in the extended longevity and an improved quality of life for humankind. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:367,412, 2005 [source] Plasma protein profiles in early asthmatic responses to inhalation allergen challengeALLERGY, Issue 1 2009T. Rhim Although mediators, such as lipids, cytokines, and chemokines, are related to the appearance of an IPR, there has been no reliable indicator to predict conditions for the appearance of an IPR. In this study, we adopted a proteomic approach to investigate the pathogenesis at the level of the plasma proteins and to develop plasma markers to predict the appearance of an IPR following an inhalation challenge with Dermatophagoides pteronyssinus (D.p.). Sixteen mild asthmatics were recruited. Plasma was obtained before challenge and when a decline in forced expiratory volume in 1 s (FEV1) values greater than 20% from the phosphate-buffered saline value was achieved during D.p. allergen challenge (positive responders), or at 60 min after the highest concentration of D.p. allergen was inhaled (negative responders). After comparing normalized volumes of the spots in the two groups, differentially expressed spots were identified using intra-gel digestion and mass spectrometric analysis. Before D.p. antigen challenge, four spots of gamma fibrinogen and its isoforms were significantly decreased and two spots of complement C3 fragments were significantly increased in the positive responders compared to the negative responders. After D.p. antigen challenge, complement C3 fragment was persistently higher, while gamma fibrinogen was lower in the positive responders than in the negative responders. A validation study using Western blotting showed that gamma fibrinogen expression in the IPR-positive asthmatics was significantly decreased compared to the average of the IPR-negative asthmatic control group. These results indicate that alterations in the complement cascade and fibrinogen may predispose patients to the appearance of an immediate response to D.p. allergen challenge and may provide plasma markers to predict the appearance of an IPR. [source] Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samplesPROTEIN SCIENCE, Issue 9 2008Stephanie M.E. Truhlar Abstract Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific 15N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific 15N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The 15N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the 15N-amino acid prevents the scrambling of the 15N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" 15N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples. [source] Quantitative proteomes and in vivo secretomes of progressive and regressive UV-induced fibrosarcoma tumor cells: Mimicking tumor microenvironment using a dermis-based cell-trapped system linked to tissue chamberPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2007Yang Shi Abstract The alterations of tumor proteome and/or in vivo secretome created by host-tumor cell interaction may be crucial factors for tumors to undergo progression or regression in a host system. Two UV-induced fibrosarcoma tumor cell lines (UV-2237 progressive cells and UV-2240 regressive cells) were used as models to address this issue. Hundreds of proteins including in vivo secretome have been identified and quantified via an isotope-coded protein label (ICPL) in conjunction with high-throughput NanoLC-LTQ MS analysis. A newly designed technology using a dermis-based cell-trapped system was employed to encapsulate and grow 3-D tumor cells. A tissue chamber inserted with a tumor cell-trapped dermis was implanted into mice to mimic the tumor microenvironment. The in vivo secretome created by host-tumor interaction was characterized from samples collected from tissue chamber fluids via ICPL labeling mass spectrometric analysis. Twenty-five proteins including 14-3-3 proteins, heat shock proteins, profilin-1, and a fragment of complement C3 with differential expression in proteomes of UV-2237 and UV-2240 cells were revealed. Three secreted proteins including myeloperoxidase, alpha-2-macroglobulin, and a vitamin D-binding protein have different abundances in the in vivo secretome in response to UV-2237 and UV-2240 cells. Differential tumor proteomes and in vivo secretome were thus accentuated as potential therapeutic targets to control tumor growth. [source] | ||||||||||||||||||||||||||||||||||