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  • 1990-1994  (13)
  • Analytical Chemistry and Spectroscopy  (9)
  • Bacteriophage T4  (2)
  • mass spectrometry
  • 1
    Electronic Resource
    Electronic Resource
    Structural analysis of bovine pancreatic thread protein (1990)
    Springer
    The protein journal 9 (1990), S. 623-632 
    Details
    ISSN: 1573-4943
    Keywords: Pancreatic thread protein ; primary structure ; mass spectrometry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Pancreatic thread protein (PTP) forms double helical threads in the neutralpH range after purification, undergoing freely reversible,pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Grosset al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing 〉99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH2-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca2+-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01025016
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  • 2
    Electronic Resource
    Electronic Resource
    Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation (1994)
    Springer
    Glycoconjugate journal 11 (1994), S. 180-186 
    Details
    ISSN: 1573-4986
    Keywords: N-terminal sequencing ; glycoprotein ; glycan analysis ; covalent immobilization ; mass spectrometry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as theO-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00731216
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  • 3
    Electronic Resource
    Electronic Resource
    Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: Analysis of mutant UvsX proteins from infected cells (1990)
    Springer
    Molecular genetics and genomics 222 (1990), S. 112-119 
    Details
    ISSN: 1617-4623
    Keywords: UvsX protein ; Bacteriophage T4
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5′ end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsX-am64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frame-shift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00283031
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  • 4
    Electronic Resource
    Electronic Resource
    Isolation and genetic characterization of newuvsW alleles of bacteriophage T4 (1990)
    Springer
    Molecular genetics and genomics 222 (1990), S. 257-264 
    Details
    ISSN: 1617-4623
    Keywords: Bacteriophage T4 ; uvsW ; DNA replication ; Recombination ; DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary TheuvsW gene of bacteriophage T4 is required for wild-type levels of recombination, for normal survival and mutagenesis after UV irradiation, and for wild-type resistance to hydroxyurea. Additionally,uvsW mutations restore the arrested DNA synthesis caused by mutations in any of several genes that block secondary initiation (recombination-primed replication, the major mode of initiation at late times), but only partially restore the reduced burst size. AuvsW deletion mutation was constructed to establish the null-allele phenotype, which is similar but not identical to the phenotype of the canonicaluvsW mutation, and to demonstrate convincingly that theuvsW gene is non-essential (althoughuvsW mutations severely compromise phage production). In an attempt to uncouple the diverse effects ofuvsW mutations, temperature-sensitiveuvsWts mutants were isolated. Recombination and replication effects were partially uncoupled in these mutants, suggesting distinct and separable roles foruvsW in the two processes. Furthermore, the restoration of DNA synthesis but not recombination in the double mutantsuvsW uvsX anduvsW uvsY prompts the hypothesis that the restored DNA synthesis is not recombinationally initiated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00633826
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  • 5
    Electronic Resource
    Electronic Resource
    Determination of an organotin stabilizer in a rigid poly(vinyl chloride) plastic by on-line supercritical fluid extraction and chromatography with formic acid modified carbon dioxide and flame ionization detection (1993)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 16 (1993), S. 198-202 
    Details
    ISSN: 0935-6304
    Keywords: Supercritical fluid chromatography ; Supercritical fluid extraction ; Poly(vinyl chloride) ; Dimethyltin mercaptide ; Formic acid ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jhrc.1240160316
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  • 6
    Electronic Resource
    Electronic Resource
    A comparison of formic acid and formamide as modifiers of supercritical carbon dioxide compatible with flame lonization detection (1993)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 16 (1993), S. 130-134 
    Details
    ISSN: 0935-6304
    Keywords: Supercritical fluid chromatography ; Packed columns ; Modifiers ; Formic acid ; Formamide ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jhrc.1240160216
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  • 7
    Electronic Resource
    Electronic Resource
    Structural characterization by mass spectrometry of native and recombinant human relaxin (1990)
    Chichester : Wiley-Blackwell
    Biological Mass Spectrometry 19 (1990), S. 655-664 
    Details
    ISSN: 1052-9306
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Mass spectrometry has played a key role in characterizing the primary structure of native and recombinant relaxin, a peptide hormone that induces ripening of the cervix prior to childbirth. The peptide is composed of two chains, A and B, and is formed from a single-chain prohormone, as is insulin. Aside from conserved cysteines, though, it has little sequence homology with insulin. Due to the small amounts of native peptide initially available ( 〈 10 pmol), traditional techniques could not provide information on the blocked A-chain sequence, on the carboxy-terminal sequences, nor on other possible post-translational modifications. Mass measurements by fast atom bombardment (FAB) were made on reduced human relaxin isolated from corpora lutea. The detection limit by FAB for reduced relaxin was 500 fmol. The B-chain was four amino acids shorter than expected from comparison of the previously known cDNA sequence with homologous rat and porcine sequences. The A-chain, as predicted, was 24 amino acids in length and had a pyroglutamic acid residue on the amino-terminus. The purified samples were homogeneous with no other post-translational modifications. The recombinant relaxin molecule was also extensively characterized by mass spectrometry. In addition to the intact molecule, all tryptic peptides were characterized by FAB. A capillary high-performance liquid chromatography continuous-flow FAB system, developed for high-sensitivity peptide mapping, aided in these analyses. Finally, the three disulfide bonds were shown by tandem mass spectrometry to match those of insulin.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bms.1200191105
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  • 8
    Electronic Resource
    Electronic Resource
    An evaluation of tandem mass spectrometry in drug metabolism studiesA preliminary account of this work was presented at the 2nd European Tandem Mass Spectrometry meeting held at the University of Warwick, UK, on 19-22 July 1992. (1993)
    Chichester : Wiley-Blackwell
    Biological Mass Spectrometry 22 (1993), S. 388-394 
    Details
    ISSN: 1052-9306
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The use of precursor ion and constant neutral loss scanning as a means of rapidly detecting drug metabolites is evaluated. Four clinically useful drugs, namely (i) cyclophosphamide, (ii) mifentidine, (iii) cimetropium bromide and (iv) haloperidol, were subjected to microsomal incubations to afford phase I metabolites. Aside from a minor clean-up procedure involving zinc sulfate precipitation of microsomal proteins and solid-phase extraction of metabolites using a Sep-pak C-18 cartridge, the mixtures were analysed directly by fast atom bombardment tandem mass spectrometry. It is demonstrated that such screening strategies are important in detecting novel metabolites. However, there are some problems associated with only using such methods, including (i) the possibility of not detecting metabolites that undergo unusual collision-induced dissociation fragmentation pathways, (ii) the non-detection of metabolites that have undergone metabolic change at unusual sites of reactivity, and (iii) production of artifacts derived from the parent drug by the primary ionization process. Examples are discussed that highlight both the strengths and weaknesses of such an approach.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bms.1200220705
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  • 9
    Electronic Resource
    Electronic Resource
    Analysis of effector cell-derived lyso platelet activating factor by electron capture negative ion mass spectrometry (1991)
    Chichester : Wiley-Blackwell
    Biological Mass Spectrometry 20 (1991), S. 545-552 
    Details
    ISSN: 1052-9306
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Quantification of 1-O-alkyl-2-lyso-sn-3-glycero-phosphocholine (lysoPAF) and determination of the different molecular species released by cells has been hampered by the molecules's lack of intrinsic bioactivity, unavailability of a suitable internal standard, and reliance on derivatives requiring electron impact techniques. We have synthesized trideuterated internal standards (labeled on the terminal carbon of the alkyl chain) for both C16:0 and C18:0 lysoPAF. Using these standards, we isolated and quantified lysoPAF released from A23187-stimulated human neutrophils and rat alveolar macrophages. Extracted lysoPAF was purified by solid-phase extraction and thin-layer chromatography. The polar phosphorylcholine group was removed with 29 M HF or phospholipase C. The two free hydroxyl groups were derivatized with pentafluorobenzoyl chloride. The resultant bis-pentafluorobenzoyl derivative, analyzed by gas chromatography/electron capture negative ion mass spectrometry, underwent substantial fragmentation. Lowering of the ion source temperature resulted in a dramatic increase in signal-to-noise ratio, with the vast majority of the ion current carried in the molecular anion. Stimulated neutrophils released 16.3 and 10.2 ng/106 cells of C16:0 lysoPAF and C18:0 lysoPAF, respectively. Rat macrophages synthesized 15.9 ng/106 cells of C16:0 lysoPAF, but C18:0 lysoPAF was variably detected at low levels. We conclude that use of the bispentafluorobenzoyl ester derivative of lysoPAF allows facile quantification of this autacoid metabolite in biological matrices.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bms.1200200907
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  • 10
    Electronic Resource
    Electronic Resource
    Importance of quality control in a gas chromatographic method to characterize crude oilsMany of the details in this paper were presented as a poster at the Tenth International Symposium on Capillary Chromatography, Riva del Garda, Italy, May 22-25, 1989. (1994)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 17 (1994), S. 463-468 
    Details
    ISSN: 0935-6304
    Keywords: Capillary GC ; Petroleum crude oil ; Condensates ; Carbon number distribution ; Pseudocomponents ; Quality control ; Quality assurance ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A quality control scheme has been developed to achieve reproducible capillary GC characterization of crude oils and petroleum condensates. The method uses an internal standard to quantify the amount of non-eluted material in the crude oil sample. Correlation between the gas chromatographic results and actual distillation was 2 %. After repeated use, however, weekly analysis of the same standard crude oil did not give the correct composition. This was a result of discrimination, which worsened with time, as a result of leaks in the septum or the graphite ferrules. More reproducible results were obtained by performing frequent analyses of quality control samples to ensure that the gas chromatographic system was operating properly. The use of quality control charts was a convenient way of ensuring correct operation and identifying the need for corrective action on the gas chromatographic system.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jhrc.1240170613
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