Fast Atom Bombardment Mass Spectrometer Research Articles

Identification of the Catalytic Residues of Carboxylesterase fromArthrobacter globiformisby Diisopropyl Fluorophosphate-Labeling and Site-Directed Mutagenesis

The role of amino acid residues in the enzymatic activity of carboxylesterase from Arthrobacter globiformis was analyzed by diisopropyl fluorophosphate (DFP) labeling and site-directed mutagenesis. The electrospray ionization mass spectrometric (ESI-MS) analysis of the esterase, covalently labeled by DFP, showed stoichiometric incorporation of the inhibitor into the enzyme. The further comparison of endopeptidase-digested fragments between native and DFP-labeled esterase by fast atom bombardment mass spectrometric (FAB-MS) analysis as well as site-directed mutagenesis indicated that Ser59 in the consensus sequence Ser-X-X-Lys, which is conserved exclusively in penicillin-binding proteins and some esterases, served as a catalytic nucleophile. In addition, the results obtained from analysis of the mutants at position 62 suggested the importance of the basic amino acid side chain at this position, and suggested the significance of this residue acting directly as a general base rather than its involvement in the maintenance of the optimum hydrogen-bonding network at the active site.

Production and Characterization of Fengycin by Indigenous Bacillus subtilis F29-3 Originating from a Potato Farm

Fengycin, a lipopeptide biosurfactant, was produced by indigenous Bacillus subtilis F29-3 isolated from a potato farm. Although inhibiting the growth of filamentous fungi, the fengycin is ineffective against yeast and bacteria. In this study, fengycin was isolated from fermentation broth of B. subtilis F29-3 via acidic precipitation (pH 2.0 with 5 N HCl) followed by purification using ultrafiltration and nanofiltration. The purified fengycin product was characterized qualitatively by using fast atom bombardment-mass spectrometer, Fourier transform infrared spectrometer, ultraviolet-visible spectrophotometer, 13C-nuclear magnetic resonance spectrometer and matrix assisted laser desorption ionization-time of flight, followed by quantitative analysis using reversed-phase HPLC system. This study also attempted to increase fengycin production by B. subtilis F29-3 in order to optimize the fermentation medium constituents. The fermentation medium composition was optimized using response surface methodology (RSM) to increase fengycin production from B. subtilis F29-3. According to results of the five-level four-factor central composite design, the composition of soybean meal, NaNO3, MnSO4·4H2O, mannitol-mannitol, soybean meal-mannitol, soybean meal-soybean meal, NaNO3-NaNO3 and MnSO4·4H2O-MnSO4·4H2O significantly affected production. The simulation model produced a coefficient of determination (R2) of 0.9043, capable of accounting for 90.43% variability of the data. Results of the steepest ascent and central composite design indicated that 26.2 g/L of mannitol, 21.9 g/L of soybean meal, 3.1 g/L of NaNO3 and 0.2 g/L of MnSO4·4H2O represented the optimal medium composition, leading to the highest production of fengycin. Furthermore, the optimization strategy increased the fengycin production from 1.2 g/L to 3.5 g/L.

Silicate species in high pH solution molybdate, whose silica concentration is determined by colorimetry

The chemical species of silica, whose concentration was measured by colorimetry, were examined in alkaline hydroxide solutions. The total amount of SiO 2 measured by colorimetry is in good agreement with that measured by weight loss on drying. The monomer (Si(OH) 3O −), monomer-Na + complex (Si (OH) 2O 2Na −), dimer (Si 2(OH) 5O 2 −), dimer-Na + complex (Si 2(OH) 4O 3Na −), linear tetramer (Si 4(OH) 9O 4 −), cyclic tetramer (Si 4(OH) 7O 5 −) and cyclic tetramer-Na + complex (Si 4(OH) 6O 6Na −) were observed in NaOH and KOH solutions by fast atom bombardment-mass spectrometer. When the NaOH solutions containing silica were neutralized, they were found to contain the monomer-Na + complex, dimer, dimer-Na + complexes (Si 2(OH) 3O 4Na 2 −, Si 2(OH) 2O 5Na 3 −) in addition to dimer-Na + complex (Si 2(OH) 4O 3Na −), linear tetramer, cyclic tetramer and cyclic tetramer-Na + complex. The solutions of silica in natural water and of silica at pH 7–12 contain monomer, monomer-Na + complex, dimer, dimer-Na + complexes, linear tetramer, cyclic tetramer and cyclic tetramer-Na + complex of silica, as determined by colorimetry with ammonium molybdate.

Asymmetric synthesis of a 3-acyltetronic acid derivative, RK-682, and formation of its calcium salt during silica gel column chromatography.

RK-682 was reported to be a potent protein tyrosine phosphatase inhibitor. We found that (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (1) was easily converted to its calcium salt during column chromatography on Silica gel 60, and this calcium salt was identical to RK-682 originally isolated from a natural source. Here we report details of the asymmetric synthesis of (R)-1 and its conversion to the calcium salt. Fast atom bombardment mass spectrometric (FAB-MS) analysis of the free and calcium salt forms of RK-682 is also reported.

Separation and characterization of peanut phospholipid molecular species using high‐performance liquid chromatography and fast atom bombardment mass spectrometry

AbstractTotal lipid extracts from peanut seed were separated on a silica column into a triacylglycerol fraction and a polar lipid fraction by high‐performance liquid chromatography (HPLC). The polar fraction containing the phospholipids was retained on the precolumn, and the triacylglycerol fraction was eluted to a waste flask by a special valve arrangement. Phospholipids were eluted from the precolumn and separated into various classes on a silica analytical column. Each phospholipid class was manually collected and subsequently subjected to reversed‐phase HPLC in tandem with a fast atom bombardment mass spectrometer. Phosphatidylethanolamine was separated into five molecular species. Phosphatidylinositol and phosphatidylcholine were each separated into six molecular species.

Determination of end groups in poly(methylmethacrylate peroxide) by fast atom bombardment mass spectrometry and IR spectroscopy

A detailed comprehensive investigation has been made to unequivocally analyse the end groups of a vinyl polyperoxide polymer namely, poly(methylmethacrylate peroxide), PMMAP, using Fast Atom Bombardment Mass Spectrometric (FABMS) technique. Further evidence to FABMS analysis has been sought from IR spectroscopic analysis on the same polymer. It has been found that PMMAP contains both hydroxyl and hydroperoxide end groups which has been explained by the chain transfer reaction of growing chain with the degradation product of PMMAP.

Occurrence and Structural Analysis of Highly Sulfated Multiantennary N‐linked Glycan Chains Derived from a Fertilization‐Associated Carbohydrate‐Rich Glycoprotein in Unfertilized Eggs of Tribolodon hakonensis

This study represents the first detailed investigation of the nature of highly sulfated (keratan-sulfate-like) complex-type asparagine-linked glycans having a tetraantennary core structure and shows the effectiveness of fast-atom-bombardment mass spectrometric (FAB-MS) methods incorporating derivatization and mild methanolysis for analyzing such complex types of sulfated glycans. The structure of the N-glycan chains was unambiguously established by a combination of compositional analysis, methylation analysis, mild methanolysis for desulfation, hydrazinolysis/nitrous acid deamination, enzymatic (endo-beta-galactosidase and peptide:N-glycosidase F) digestions, and instrumental analyses (1H-NMR spectroscopy and FAB-MS) which revealed the novel repeating sulfated carbohydrate sequences, +/- Gal beta 1-->4Gal beta 1[-->(HSO3-->6)GlcNAc beta 1-->3(+/- Gal beta 1-->4)Gal beta 1]n--> (see Structure I; p + q + r + s approximately 14). This sequence is unique in: (a) the skeletal structure is similar to that of keratan sulfate but is completely devoid of 6-O-sulfated Gal residues and (b) the presence of branched Gal residues in the sequence -->4GlcNAc beta 1-->3(Gal beta 1-->4)Gal beta 1-->. [formula: see text]

Human α‐fetoprotein produced from hep G2 cell line: Structure and heterogeneity of the oligosaccharide moiety

AbstractThe carbohydrate moiety of human α‐fetoprotein, an RMM 67000 glycoprotein produced in a hepatoma cell line (Hep G2), was investigated by the combined use of high‐resolution chromatographic techniques and mass spectrometry. Fast atom bombardment mass spectrometric (FABMS) and reversed‐phase high‐performance liquid chromatographic analysis of α‐fetoprotein obtained from a large‐scale cell culture following tryptic and peptide N‐glycanase F hydrolysis demonstrated that the protein contains a single glycosylation site at level of asparagine 232. Further, electrospray mass spectrometric measurement of the intact protein molecular mass showed that two main glycoforms are present. The complete definition of the structural heterogeneity of the oligosaccharide moiety was achieved by high‐performance anion‐exchange chromatography with pulsed amperometric detection together with carbohydrate mapping by FABMS of the released oligosaccharides demonstrating (i) that the glycosylation produced by cell culture is of the biantennary complex type typical of human hepatoma α‐fetoprotein and (ii) the presence of two main structures in a ratio of about 2:1 differing in the presence of a fucose residue in the N‐acetylglucosamine in the non reducing portion of the molecule.

Chemical synthesis and biological activity of the EGF-like domain of heparin-binding epidermal growth factor-like growth factor (HB-EGF).

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently discovered member of the epidermal growth factor (EGF) family. This novel growth factor possesses the EGF-like domain in the carboxyl portion. In order to evaluate the biological function of the EGF-like domain in HB-EGF, human HB-EGF(44-86) corresponding to the EGF-like domain was synthesized by the solid-phase procedure using the Fmoc strategy. It was confirmed by amino acid microsequencing of cysteine-containing fragments derived from thermolytic digestion that the pattern of three disulfide bond pairings in synthetic HB-EGF(44-86) was consistent with that of EGF and transforming growth factor-alpha (TGF-alpha). The homogeneity of the synthetic peptide was confirmed by analytical RP-HPLC, amino acid analysis and fast atom bombardment mass spectrometer (FAB-MS). Compared with h-EGF, the EGF-like domain of human HB-EGF showed a comparable mitogenic activity in the proliferation of NIH/3T3 fibroblast cells. These results suggest that the EGF-like domain of human HB-EGF may play an important role in mitogenic activity.

Improvement of Chemical Analysis of Antibiotics. Part XIX1: Determination of Tetracycline Antibiotics in Milk by Liquid Chromatography and Thin-Layer Chromatography/Fast Atom Bombardment Mass Spectrometry

A precise liquid chromatographic (LC) determination with high sensitivity and a reliable mass spectrometric (MS) confirmation were established for the determination of tetracycline antibiotics (TCs) in milk. The determination of the TCs was based on C18 cartridge cleanup followed by LC separation. Recoveries of TCs from milk fortified at 20 ppb were 73.0-81.8%, and coefficients of variation were 2.6-4.6%. With this method, concentrations of oxytetracycline and tetracycline as low as 10 ppb and chlortetracycline and doxycycline as low as 20 ppb were determined readily. Thin-layer chromatography (TLC)/fast atom bombardment (FAB) MS with a condensation technique was used to confirm TCs in milk. After the C18 cartridge cleanup, separation on a C8 TLC plate, and application of the sample condensation technique, FAB mass spectra were measured. TCs in milk at 50 ppb were reliably confirmed by TLC/FABMS.

Paralytic shellfish poisons from Australian cyanobacterial blooms

Saxitoxin-group neurotoxins (paralytic shellfish poisons) have been identified in a cultured strain of Anabaena circinalis and in natural bloom samples in which this species was the dominant organism collected from widely distributed sites in the Murray-Darling Basin of Australia. These toxins have hitherto been isolated almost exclusively from 'red tide' dinoflagellates and contaminated shellfish. Two 'aphantoxins', which appear to be identical to two of the paralytic shellfish poisons, have been identified in a cyanobacterium from a small number of sites in New Hampshire, USA. The conclusions are supported by electrophysiological studies and by high-performance liquid chromatographic (HPLC) and fast atom bombardment-mass spectrometric (FAB-MS) analyses.

Relative metal ion affinities of organic nitriles in the gas phase

AbstractThe relative metal ion (Ni+ and Co+) affinities of 14 alkanenitriles, alkenenitriles and benzonitrile were estimated using Cooks' kinetic method in a fast atom bombardment mass spectrometer. The results are compared with proton affinities, affinities for other metal ions, two‐ligand dissociation enthalpies and the dipole moments of the nitriles. The RCNCo+ bond is found to be close to the RCNNi+ bond but weaker than the RCNAl+ bond. The effective temperature (T) of the metal‐bound dimer ions falls in the range 298 K < T < 400 K.

Marine biosurfactants. IV. Production, characterization and biosynthesis of an anionic glucose lipid from the marine bacterial strain MM1

During screening for biosurfactants among marine, n-alkane-utilizing bacteria, low- and high-molecular surface-active substances were detected. The marine bacterial strain MM1 was found to synthesize a novel glycolipid that has not so far been cited in the literature. Both 1H, 13C-nuclear magnetic resonance spectroscopic and positive ion fast atom bombardment mass spectrometer studies led to the identification of a glucose lipid consisting of four 3-OH-decanoic acids, which are linked together by ester bonds. The lipophilic moiety is coupled glycosidically with C-1 of glucose. The glucolipid reduced the surface tension from 72 mN/m to 30 mN/m while the minimum interfacial tension towards n-hexadecane was lowered to values smaller than 5 mN/m.

Detection of benzo[ a]pyrene sulfate and glucuronide conjugates in cell culture medium by directly coupled microbore high-performance liquid chromatography—fast atom bombardment mass spectrometry

An improved method is described for detecting glucuronide and sulfate conjugates of benzo[ a]pyrene in medium from cell cultures treated with benzo[ a]pyrene. This method is based on a microbore high-performance liquid chromatograph directly coupled to a high-resolution continuous-flow fast atom bombardment mass spectrometer. Sulfate and glucuronide conjugates, as well as some structural isomers of glucuronide conjugates, were fully separated by the reversed-phase microbore high-performance liquid chromatography conditions used in this study. Since the method does not rely on the use of radiolabeled materials, it may be used to detect conjugates of a wide variety of hydrocarbons. The high sensitivity and selectivity of the method were demonstrated by detecting conjugates in the media of cell cultures derived from mice, hamsters and humans.

Human .alpha.-fetoprotein primary structure: a mass spectrometric study

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.

Optimization of Chromatographic Conditions for Combined Micropore HPLC/Continuous-Flow Fast-Atom Bombardment Mass Spectrometry

System parameters for microbore high-performance liquid chromatography (HPLC) of proteolytic digests are optimized to provide chromatographic separations amenable to the flow rates (5 to 10 µL/min) used with the continuous-flow fast-atom bombardment mass spectrometer (MS) interface. Results that show the effects of column dimensions, the rate at which solvent gradients are delivered to the microbore column, column flow rates, and the slopes of eluting solvent gradients on these separations are presented. Optimal conditions are achieved using a 1 × 50-mm column at volumetric flow rates of 20 to 25 µL/min combined with precolumn flow splitting that minimizes the delay time for delivery of solvent gradients to the column. Postcolumn splitting (1:4) is used to give optimal interface flow rates and to provide simultaneous MS and UV detection. The LC/MS analysis of the tryptic digest of human apolipoprotein A-I is shown to illustrate the utility of the methods.

Rapid characterization of natural and biotechnologically synthesized human growth hormones by fast atom bombardment mass spectrometry and high-performance liquid chromatography.

Fast atom bombardment mass spectrometric (FAB-MS) analysis of tryptic digest of human growth hormone (hGH) was caried out to verify the primary structures of two kinds of hGHs expressed in Escherichia coli; native type (r-hGH) and N-terminal methionine-containing type of hGH (met-hGH. Both structures were confirmed by comparing the mass spectra of the tryptic digest mixtures with that of authentic hGH extracted from human pituitary (p-hGH). The signals detected in the mass spectura accounted for 82% of the sequence of hGH. In order to determine the location of disulfide bonds, a method was designd for selective preparation of peptide fragments containing a disulfide bond from the tryptic digest. The selection was performed by comparison with high-performance liquid chromatograms before and after the reduction of the digest mixtures with dithiothreitol (DTT). From the mass spectra of two disulfide-containing peptide fragments, the locations of two disulfide bonds in the hGH sequence were confirmed to be the same among the three types of hGHs. The present study indicates that the FAB-MS provides an excellent method for the rapid verification of the primary structure of proteins, and the combination of high-performance liquid chromatography (HPLC) with this method facilitates and analysis of disulfide bonds.

Fluorescent labeling of cysteinyl residues. Application to extensive primary structure analysis of proteins on a microscale.

The specificity and efficiency of fluorescent labeling of proteins by reduction and subsequent alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) [J.J. Gorman, (1987) Anal. Biochem. 160, 376-387] has been investigated. Proteins studied include porcine insulin, chicken ovalbumin and bovine serum albumin. Amino acid analysis of the B-chain derivative of insulin revealed quantitative recovery of cysteine in its S-carboxymethyl form and no other carboxymethylated amino acid derivatives. Fast-atom-bombardment mass spectrometric (FAB-MS) analysis of this derivative also indicated specific labeling of cysteine residues and automated stepwise protein sequence analysis of the derivative was performed to completion with initial and average repetitive yields of 73% and 96%, respectively. Tryptic peptides produced from the ovalbumin and serum albumin derivatives were fractionated by HPLC and subsequently analysed by amino acid analysis, FAB-MS and automated stepwise protein sequence analysis. These analyses have revealed that the labeling procedure exhibits a high degree of efficiency and is specifically directed towards S-alkylation of cysteine residues. The high level of fluorescence intensity of the label enabled specific detection of trace quantities of cysteine-containing peptides derived from contaminating protein(s). It is apparent that in addition to facilitating isolation of small quantities of proteins the labeling procedure is compatible with standard protein chemistry techniques involved in obtaining extensive structural data on isolated proteins.

The Use of Fast Atom Bombardment (FAB) Mass Spectrometry for Monitoring Petrochemicals

Abstract In recent years ionisation by Fast Atom Bombardment (FAB) has been developed to enable mass spectrometric (MS) analysis to be performed on samples containing polar, involatile compounds without any pre-derivatisation. This makes FAB MS an ideal candidate for the analysis of polar petrochemical waste products. In addition, the soft ionisation induced by FAB minimises fragmentation, enabling complex mixtures to be screened routinely without lengthy sample work-up. Confirmatory identification of components can be obtained at high MS resolution. The present study reports the use of FAB MS to monitor specific petrochemicals in the aqueous effluents from certain coastal plants.

Negative ion fast atom bombardment mass spectrometry.In situreactions of boronic acids with triols and related compounds, sugars and nucleosides

On the probe tip of a fast atom bombardment mass spectrometer, boronic acids react with a wide range of trifunctional compounds of appropriate configuration yielding negatively-charged boronate cage compounds. Formation of 5- to 8-membered rings occurs readily in the mass spectrometer. The reaction constitutes a simple and effective means of pre-ionizing boronic acids, triols, aminodiols, monomercaptodiols and related compounds including sugars and nucleosides. The resulting boronate complexes exhibit excellent negative ion fast atom bombardment mass spectra, indicating wide application for (1) analysing polyhydroxy compounds and boronic acids, (2) providing information on configuration of polyhydroxy compounds, and (3) measuring affinity constants of polyfunctional compounds for boronic acids. The use of polyethylene glycol as solvent and mass calibrant is also discussed.