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Fast atom bombardment mass spectrometry that can directly analyze lysophospholipids was used to quantitatively determine the kinetics of phospholipase A2. This method is 1250 times more sensitive than the colorimetric assay.
Fast atom bombardment mass spectrometry has already been used for the identification of mutations in abnormal human hemoglobin chains. This paper presents new results obtained with this technique. The methodology used here is compared with more conventional biochemical techniques and automated microsequencing. In every case, a well-chosen combination of peptide-high performance liquid chromatography, mass spectrometry, amino acid analysis, and sequence analysis led rapidly to the identification of the mutant. The high sensitivity of these techniques holds great promise for the analysis of molecular abnormalities in various genetic disorders presently detectable only by the application of a molecular biological approach.
Fast atom bombardment mass spectral mapping of endoproteinase Asp-N digest mixtures is used for characterization of new synthetic linear and cyclic glucagon analogs. The results allow rapid identification of sequence modifications in linear glucagon analogs. For the cyclic compounds, the technique allows confirmation of the presence and position of the cyclic amide bond, as well as verification of the sequence of the modified glucagon analogs. The specificity of the Asp-N enables differentiation of isometric glucagon analogs which differ only in the position of the cyclic amide bond. Important information concerning the purity of the synthetic analogs is also available.
Fast atom bombardment mass spectrometry (FABMS) was applied to the direct detection of silica species dissolved in LiCl, NaCl, MgCl(2), CaCl(2) and SrCl(2) solutions in order to investigate its dissolution process in solution. Several species of dissolved silicate complexes in the solution were directly detected by FABMS. The peak intensities of [SiO(2)(OH)(2)Na](-), [SiO(3)(OH)Ca](-) and [SiO(3)(OH)Sr](-) increased with increasing concentrations of NaCl, CaCl(2) and SrCl(2), whereas the peak intensities of [SiO(2)(OH)(2)Li](-) and [SiO(3)(OH)Mg](-) did not increase with increasing concentrations of LiCl and MgCl(2). These results indicte that silicate and cation bind in the solution not after but before ionization. The isotope pattern of Sr(2+) confirmed the existence of the silicate-Sr complex not only with increase of the concentration of silica but also the mass numbers of Sr. The silicate complexes formed with Na(+), Ca(2+) and Sr(2+) showed high stability in chloride solution. This is in good accordance with the fact that Na(+), Ca(2+) and Sr(2+) accelerate the dissolution of silica to form complexes during solution equilibrium. Considering that the stability constant was examined and reported in other papers, this new findings that Mg(2+) does not form a complex with silicic acid (Si(OH)(4)) is very important.
Fast atom bombardment mass spectrometry has been applied to the localization of phosphorylation sites in buffalo beta-casein. Two complementary strategies of identification are described. Phosphorylated residues in the tryptic peptide Tp 1 have been assigned by measuring the masses of peptide fragments obtained by enzymatic degradations. The phosphoserine residue in peptide Tp 2 has been identified by determining the intact molecular weight and confirmed by partial sequence information. This rapid and sensitive procedure appears of a great interest in structural studies of a wide range of post-translational modifications in proteins.
Fast atom bombardment mass spectrometry (FAB MS) and fast atom bombardment mass spectrometry-mass spectrometry (FAB MS/MS) were used to study the monovalent glycoside polyether antibiotics maduramicin alpha, beta and delta and the maduramicin alpha salts, their derivatives and degradation products. Also, representative compounds from three major classes of polyether antibiotics were studied: the monovalent polyethers, nigericin and monensin A, the divalent polyether lasalocid A and the monovalent glycoside polyethers septamycin, BL580 delta, etheromycin and carriomycin. The respective FAB fragment and decomposition ions were correlated with the known structures. The FAB spectra of all the polyethers contained metal-adduct molecular ions. Protonated molecular ions were absent. All the polyethers having a beta-hemiketal carboxylic acid group produced an abundant ion, often the base peak of the spectra, 62 daltons less than the corresponding metal-adduct molecular ion. The gas phase mechanism proposed for the formation of this fragment ion is an unusual unimolecular reaction which is initiated by an intramolecular proton transfer from the carboxylic acid to the hydroxy group of the beta-hemiketal, and, then followed by the concerted losses of water and carbon dioxide to produce the corresponding polyether olefin.
Fast atom bombardment (FAB) mass spectrometry was employed to identify and quantitate dansyl amino acids obtained in the N-terminal analysis of proteins. FAB mass spectra of dansyl amino acids are characterized by intense quasimolecular ions formed by cationization of the molecular species and fragment ions produced by cleavage of the bonds on either side of the sulfanyl group. Investigation of the dansyl amino acid responses using dansyl aminobutyric acid as an internal standard showed that dansyl amino acids can be determined quantitatively at a level of 0.1 nmol. N-terminal residue analysis was performed on a number of proteins to substantiate the technique including bovine serum albumin, pepsinogen, trypsinogen, bromelain, ribonuclease A, and bacteriophage P-22 tail protein.
Fast atom bombardment (FAB) mass and MS/MS spectra of a novel quinone antibiotic are presented. Their interpretation is based upon the examination of reductive behavior of model quinones in FAB solvents.
Three glutathione conjugates of acetaminophen were characterized by fast atom bombardment/mass spectrometry (FAB/MS) and fast atom bombardment/mass spectrometry/mass spectrometry (FAB/MS/MS). The conjugates, 3-(glutathion-S-yl)acetaminophen, 3-(glutathion-S-yl)diacetaminophen and 3-(diglutathion-S-yl)diacetaminophen showed intense [MH]+ ions at m/z 457, 606 and 911, respectively. Only 3-(glutathion-S-yl)acetaminophen showed any fragmentation by FAB/MS. Structurally characteristic fragmentation was observed with all three conjugates when the [MH]+ ions were collisionally activated. The loss of the glycine (GLY) and glutamic acid (GLU) moieties indicated the presence of at least one glutathione (GSH) group. Multiple losses, some of which could only occur via cleavages in both GSH moieties, were observed with the diglutathione conjugate.
Tris(isopropylphenyl/phenyl) phosphates and triethylene glycol dicaprate/caprylate mixtures of typical industrial compositions have been analyzed by fast atom bombardment (FAB) mass spectrometry, FAB collisional activation (CA) mass-analyzed ion kinetic energy spectroscopy and FAB/electron impact (EI) CA B/E linked scans. Data obtained by these state-of-the-art methodologies are compared qualitatively and semi-quantitatively with those provided by classical gas chromatography (GC)/EI MS. To identify the plasticizers in cases of suspected contamination, GC/EI MS still appears to be the method of choice whenever ultimate sensitivity and specificity are needed. In cases where these requirements are less demanding, FAB mass spectrometry and FAB tandem mass spectrometry may be sufficient and more convenient.
We have examined the synthetic N-oxides of five ethylenediamine-type antihistamines using fast atom bombardment (FAB) mass spectrometry and FAB tandem mass spectrometry (MS/MS). Fragmentation of the protonated molecule in the normal and collisionally activated spectra appeared to be characteristic for this class of antihistamine N-oxide. Spectra were also acquired from an ethanolamine and a propylamine antihistamine N-oxide for comparison. These results were very similar to those obtained from biologically produced antihistamine N-oxides, as well as isomeric metabolites, which were readily distinguished from the N-oxides by characteristic fragmentation. In addition, a prominent ion 16 daltons lower in mass, which has been attributed to loss of elemental oxygen from the protonated N-oxide in chemical ionization mass spectral studies, was shown to be a matrix-dependent product of the solution-phase reduction of the antihistamine N-oxide to the parent antihistamine during FAB ionization. These results demonstrate that with a non-reducing matrix such as glycerol, FAB mass spectrometry and FAB MS/MS are excellent methods for the characterization of the non-conjugated antihistamine metabolites such as the N-oxides.
The characterization of two abnormal human haemoglobins by fast atom bombardment (FAB) mapping is presented. The first variant, called 'R', exhibits a tryptic FAB map identical to that of normal haemoglobin. However, using Staphylococcus protease V8, a peptide containing the carboxyl end of the beta-chain exhibits a mass shift down to 300 mass units. This clearly indicates the deletion of the two last amino acids of the beta-chain. The second variant, called 'Grenoble', is due to two different modifications of the beta-chain. The location of the Pro----Ser exchange on peptide T5 is achieved by the collisionally activated dissociation mass analyzed ion kinetic energy spectra of the corresponding [MH]+ ion. The m/z value of that peptide indicated a supplementary acid----amide modification, which was located by amino acid sequencing using chemical methods. This work concludes with the necessity of using complementary methods for achieving rapid determinations of abnormal proteins with minute amounts.
Fast-atom bombardment mass spectrometry was used to follow the time course of disulfide bond formation during in vitro refolding of recombinant human macrophage-colony-stimulating factor. The content of iodoacetamide-alkylated half-cystines in proteolytic peptides of trapped refolding intermediates collected at 0, 6, 17, 24, and 72 hr was determined under reducing conditions. Size-exclusion high-performance liquid chromatography analyses of the collected alkylated samples indicate that aggregated monomer proceeded through a nonaggregated monomer to an intermediate dimer and finally to the fully folded and active dimer. Underalkylation was first detected by fast-atom bombardment mass spectrometry in 17-hr samples at Cys157 and Cys159 and this corresponded to the first sample containing dimer. Analyses of intermediates from subsequent time points indicated a decrease in alkylated sulfhydryls, and at 72 hr no alkylated peptide was detected. Early samples containing only monomer showed no evidence of disulfide bonds, and the occurrence of disulfide shuffling at the monomer stage could be ruled out under the highly reducing conditions used for refolding. Biological activity was not detectable in early samples but increased to 3.6% after 24 hr of refolding and to 86% of maximum at the 72-hr time point.
Molecular size information for polymerized catechin larger than the decamer in unripe apple was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by fast-atom bombardment mass spectrometry. Matrix-assisted laser desorption/ionization time of flight mass spectrometry provided evidence for the pentadecamer using trans-3-indoleacrylic acid as the matrix in the presence of silver ion. Even in the absence of silver ion, the dodecamer and undecamer were observed in the positive- and negative-ion modes, respectively. Fast-atom bombardment mass spectrometry also afforded evidence for the undecamer in both positive- and negative-ion modes.
Fast atom bombardment/mass spectrometry or liquid secondary ion mass spectrometry provides the capability for direct analysis of steroid conjugates (sulfates, glucuronides) without prior hydrolysis or derivatization. During the analysis of biologic extracts, limitations on the sensitivity of detection arise from the presence of co-extracted material which may suppress or obscure the analyte signal. A procedure is described for the quantitative determination of dehydroepiandrosterone sulfate in serum which achieved selective isolation of the analyte using immunoadsorption extraction and highly specific detection using tandem mass spectrometry. A stable isotope-labeled analog [( 2H2]dehydroepiandrosterone sulfate) was used as internal standard. Fast atom bombardment of dehydroepiandrosterone sulfate yielded abundant [M-H]- ions that fragmented following collisional activation to give HSO4-; m/z 97. During fast atom bombardment/tandem mass spectrometry of serum extracts, a scan of precursor ions fragmenting to give m/z 97 detected dehydroepiandrosterone sulfate and the [2H2]-labeled analog with a selectivity markedly superior to that observed using conventional mass spectrometry detection. Satisfactory agreement was observed between quantitative data obtained in this way and data obtained by gas chromatography/mass spectrometry of the heptafluorobutyrates of dehydroepiandrosterone sulfate and [2H2]dehydroepiandrosterone sulfate obtained by direct derivatization.
Fast-atom-bombardment mass spectrometry has been applied to acetylated neutral and phosphorylated oligosaccharides from yeast glycoproteins and to their acetolysis products. Although acetylation increases the sample molecular weight and the complexity of the spectra, it also enhances the sensitivity of detection, is applicable to samples that contain salt, and is especially useful for analysis of phosphorylated derivatives. Acetylation by trifluoroacetic anhydride/glacial acetic acid is particularly convenient and can be done rapidly on a small amount of material. Acetolysis by acetic anhydride/glacial acetic acid/H2SO4 is done on the acetylated oligosaccharides, and the acetylated fragments are recovered by solvent extraction and immediately subjected to mass spectrometry. The methodology allows molecular weight determinations and sequence analysis by acetolysis to be carried out on a few micrograms of isolated oligosaccharide in a few hours.
Fast atom bombardment-mass spectrometry (FAB-MS) was used to analyse lipid extracts of bacteria to assess its usefulness for analysing anionic phospholipids of potential chemotaxonomic value. The following micro-organisms were tested: Acinetobacter calcoaceticus, Acinetobacter sp., Citrobacter freundii, Enterobacter cloacae (2 strains), Escherichia coli (3 strains), Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Plesiomonas shigelloides, Proteus mirabilis (3 strains), Serratia liquefaciens and Serratia marcescens. Negative-ion spectra provide data for twenty-seven major carboxylate anions (m/z 209-325) and for thirty-seven major phospholipid anions (m/z 645-774). Generally, the largest carboxylate peaks were due to 16:1, 16:0, cyc17 and 18:1 while the largest phospholipid anion peaks were due to PE(32:1), PE(33:1), PE(34:1), PE(34:2), PG(30:2), PG(31:2), PG(32:2), PG(34:1) and PS(33:0). However, quantitative differences were observed. For example, Acinetobacter lacked PE (33:1) but had exceptionally high peaks at m/z 748, PS(33:0), and m/z 281, octadecanoate. Unknown 'carboxylate' peaks were detected at m/z 254, 256, 261, 268, 282 and 301. In some cases, unknown peaks appeared to constitute possible homologous series being separated by delta m/z of 14(identical to methylene). For chemotaxonomic purposes, the complexity of the data required numerical analysis. Using the Pearson coefficient of linear correlation, as a measure of association, it was possible to compare all strains analysed. Typical results for strain comparisons were as follows: Ent. cloacae vs Ent. cloacae, r = 0.90 (Ent. cloacae vs Ac. calcoaceticus, r = 0.46). Thus FAB-MS represents an excellent means of obtaining large quantities of data on polar lipids of a range of bacterial isolates, which may be suitable for chemotaxonomic purposes.
The fast atom bombardment (glycerol) and 252Cf plasma desorption mass spectra of 16 simple monoglucosyl conjugates have been compared. Plasma desorption mass spectrometry, in general, has been found to be the superior technique for characterizing these low molecular weight conjugates because of the relative absence of interfering matrix peaks.