Positive Fast Atom Bombardment Research Articles

Differentiation of isomeric conjugated bile acids using positive-ion B/E linked scans

The main objective of this work was to use positive-ion fast atom bombardment mass spectrometry (FAB-MS) B/E linked scan spectra to investigate the possibility of differentiating positional isomers of various authentic glycine- and taurine-conjugated bile acids. Sodium salts of 14 conjugated bile acids were individually ionized by FAB-MS and characterized by scanning simultaneously the magnetic field B and the electric sector field E such that B/E remained constant throughout the scan. The dominant fragment ions could be related to cleavage of the aliphatic side chain with charge retention on the conjugated end of the bile acids. However, fragment ions arising from ring cleavages were also observed and could be used to distinguish the positions of substituent hydroxyl groups. For example, ring cleavage of conjugated dihydroxy bile acids at C-7/C-8 and C-9/C-10 permitted the differentiation of chenodeoxycholyltaurine (3 alpha,7 alpha-substitution pattern) from deoxycholyltaurine (3 alpha,12 alpha-substitution pattern) based on the presence of fragment ions at m/z 388 or m/z 404, which were indicative of hydroxyl group substitutions at either the 7- or 12-positions, respectively. It was concluded that B/E linked scans can be used to discriminate positional isomers of conjugated bile acids.

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H] + ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.

Rhizoferrin ? a novel siderophore from the fungusRhizopus microsporus var.rhizopodiformis

From a strain ofRhizopus microsporus var.rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HCl and subsequent gas chromatography/mass spectrometry (GUMS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmedN 1,N 4-bis(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia ofR. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.

Characterization of Sanguinaria canadensis L. fluid extract by FAB mass spectrometry

Positive-ion fast atom bombardment mass spectrometry was used for the rapid characterization of commercial Sanguinaria canadensis L. fluid extracts. Quaternary and non-quaternary benzophenanthridine alkaloids afford persistent peaks due to [M]+ and [M+H]+ ionic species, respectively, and their relative abundances are in good agreement with previously reported per cent analytical data. The procedure allowed sanguinarine, chelerythrine, chelirubine, sanguilutine, protopine, allocryptopine and the isomers sanguirubine and/or chelilutine to be effectively detected by means of persistent and intense peaks in all the samples examined.

Positive ion fast atom bombardment mass spectrometric analysis of the molecular species of glycerophosphatidylserine

The structure of 1,2-dipalmitoyl- sn-glycero-3-phosphoserine was analyzed by positive ion fast atom bombardment mass spectrometry and collisional activation mass-analyzed ion kinetic energy spectroscopy. The molecular weight, the polar-head group, and the fatty acid composition of this species were identified by the appearance of protonated and solvated protonated species ions, diglyceride and monoglyceride fragment ions. After purification of glycerophosphatidylserine from bovine brain and rat kidney by normal phase high-performance liquid chromatography, molecular species were identified by either positive or negative ion fast atom bombardment mass spectrometry. The study suggests that negative ion fast atom bombardment ionization is a more powerful tool for the identification of the molecular species of glycerophosphatidylserine from biological samples. Positive ion fast atom bombardment represents a useful alternative for analysis of major molecular species in natural glycerophosphatidylserine.

Fast atom bombardment ionization of porphyrins: studies of reduction processes and use in coupling of thin-layer chromatography with mass spectrometry

The positive-ion fast atom bombardment mass spectra of porphyrins usually contain substantial signals corresponding to ions from the reduced forms of the protonated molecule, specifically those ions that incorporate extra hydrogen atoms. Occurrence of such reduction reactions is not unexpected since the same processes have been throughly characterized in ammonia chemical ionization. Several authors have also noted the presence of these ions in fast atom bombardment mass spectra. However, this work documents the effect of several experimental parameters on the extent of these reactions.

Differentiation of Isomeric Purine and Pyrimidine Mononucleotides by Fast Atom Bombardment Tandem Mass Spectrometry

Abstract The use of positive ion fast atom bombardment mass-analysed ion kinetic energy (FAB/MIKE) spectroscopy to differentiate the 2′, 3′-and 5′-monophosphate isomers of adenosine, guanosine and cytidine is described.

Identification of phosphoramide mustard/DNA adducts using tandem mass spectrometry

The reaction pathway of alkylating agents is often exploited in the design of bifunctional anti-cancer drugs. These drugs form mono-DNA adducts as well as inter- and intra-strand cross-linked adducts, notably by reaction at DNA bases, including the N-7-position of guanine (G). A positive-ion fast-atom bombardment (FAB) mass spectrum of an in vitro preparation of DNA alkylated with phosphoramide mustard (the active metabolite of the anti-cancer drug cyclophosphamide) indicated the presence of the two mono-DNA adducts N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G, and N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G-OH, (MH+ 257/259 and 239, respectively) but not the presence of the cross-linked adduct N,N-bis-[2-(7-guaninyl)ethyl] amine, designated G-NOR-G (MH+ 372). Using synthetic standards, daughter-ion spectra of NOR-G, NOR-G-OH and G-NOR-G were obtained (matrix 0.2 M p-toluene sulphonic acid in glycerol) by positive-ion FAB tandem mass spectrometry (FAB-MS/MS). The daughter-ion spectra of both mono-DNA adducts NOR-G and NOR-G-OH contained a fragment ion at m/z 152 [G + H]+, whereas the cross-linked adduct, G-NOR-G, showed an ion at m/z 221, [MH-G]+. Evidence for the presence of NOR-G, NOR-G-OH and G-NOR-G in the in vitro preparation was obtained by performing a double parent-ion scan on m/z 152 and 221. The presence of G-NOR-G was further supported by performing a single parent-ion scan on m/z 221.(ABSTRACT TRUNCATED AT 250 WORDS)

FAB-MS/MS Spectrometry in Determining the Primary Structure ofγ-Glutamyl-Containing Peptides

Positive fast atom bombardment tandem mass spectrometry (FAB-MS/MS) was applied for peptide sequencing, particularly for determining the gamma glutamyl linkage involved in metal-binding peptides such as Cadystin (γEC)3G = Cadystin A and Cadystin (γEC)2G = Cadystin B (MW 771 and 539, respectively). The fragmentation patterns between the natural gamma glutamyl peptide and the synthetic alpha glutamyl one were clearly distinguishable. FAB-MS/MS was proved to be a good method for determining these peptides, since it needed no chemical degradation and only a small amount of the peptide was needed for determination.

Structural characterization of monophosphoryl lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide.

Sixteen monophosphoryl Lipid A (MLA) homologs obtained from the lipopolysaccharides of Salmonella minnesota Re595 were separated by preparative thin layer chromatography into eight fractions. The components of these fractions were analyzed directly (or as structural analogs) and characterized by mass spectrometry. Molecular weights were determined by negative and positive ion fast atom bombardment mass spectrometry and component structures were assigned following a study of fragmentation and metastable ion kinetic energy spectrometry. One fraction (TLC-8) contained a single heptaacyl MLA of Mr = 1,954, a structure previously elucidated (Qureshi, N., Mascagni, P., Ribi, E., and Takayama, K. (1985) J. Biol. Chem. 260, 5271-5278). The remaining seven fractions contained 15 additional MLAs with decreasing acylation. Two of these components have been previously reported in S. minnesota and Salmonella typhimurium. Three of the eight TLC fractions (TLC-8, -7, -6) were found to be biologically active toward human platelets inducing their aggregation and secretion of serotonin. All tested fractions induced varying degrees of phosphorylation of a platelet protein of Mr = 47,000 (P47) reflecting protein kinase C activation (Grabarek, J., Her, G. R., Reinhold, V. N., and Hawiger, J. J. (1990) J. Biol. Chem. 265, 8117-8121).

Strategies for characterization of ganglioside inner esters I—fast atom bombardment mass spectrometry

A comparison of old and new fast atom bombardment (FAB) mass spectrometric strategies for characterization of ganglioside inner esters (lactones) is presented. Data obtained for lactones of GD3 (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) using negative ion FAB mass spectrometry of underivatized materials, negative ion FAB mass spectrometry following ammonolysis, and positive ion FAB mass spectrometry following ammonolysis and permethylation are presented and discussed. The latter method uses well-known reactions to produce a novel type of ganglioside derivative, highly amenable to analysis by positive ion FAB mass spectrometry, which is introduced to simplify unambiguous location of NeuAc residues involved in ester linkages to other sugars.

Characterization of glutathione conjugates of chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry

Chlorambucil (p-(di-2-chloroethyl)amino-gamma-phenylbutyric acid) is a bifunctional alkylating agent which exhibits acquired drug resistance upon repeated dosing in humans. This compound reacts with glutathione both non-enzymatically and enzymatically in the presence of immobilized microsomal glutathione-S-transferases to produce several glutathione conjugates. These conjugates result from displacement of one or both chlorines by the nucleophilic cysteine sulfhydryl moiety of glutathione. The mono- and diglutathionyl conjugates of chlorambucil were purified by reversed-phase high-performance liquid chromatography and characterized by positive ion fast atom bombardment mass spectrometry. In addition, the mono- and dihydroxy hydrolysis products of chlorambucil were characterized by positive ion thermospray liquid chromatography/mass spectrometry (LC/MS). The glutathione conjugates of chlorambucil did not produce molecular ion species in thermospray LC/MS mode, but gave characteristic ions at m/z 147 corresponding to fragmentation of the glutathione moiety. The formation of glutathione conjugates of this class of alkylating agents may play a role in the development of acquired drug resistance.

Formylated peptides from cyanogen bromide digests identified by fast atom bombardment mass spectrometry

Exposure of proteins to 70% formic acid during cyanogen bromide digestion can result in formation of artifact peaks during subsequent purification by HPLC and detection of [M + H + 28] + ions during analysis by fast atom bombardment (FAB) mass spectrometry. Following cyanogen bromide digestion, peptides from equine heart cytochrome c, bacteriorhodopsin, bovine adrenal medulla dodecapeptide, and bovine adrenal peptide E were analyzed by positive ion FAB mass spectrometry. The cyanogen bromide peptides of cytochrome c and bacteriorhodopsin showed mixtures of formylated ions, [M + H + 28] +, and nonformylated ions, [M + H] +. Bovine adrenal medulla dodecapeptide and bovine adrenal peptide E were not formylated during digestion. Formylated peptides could be resolved from the corresponding nonformylated peptides using reversed-phase HPLC. Instability of the formylated peptides prevented localization of the adduct by Edman degradation. However, B E - linked scanning during FAB mass spectrometric analysis with collisional activation of the [M + H + 28] + ion of a cyanogen bromide peptide from cytochrome c suggested that formylation occurred at a threonine residue. On the basis of stability measurements in aqueous solution and analysis by FAB mass spectrometry, it was determined that serine and threonine residues are the most likely sites of esterification by formic acid during cyanogen bromide digestion of proteins. Furthermore, substitution of 70% trifluoroacetic acid for formic acid during cyanogen bromide digestion eliminated formylation and generated little or no trifluoroacetylation.

Use of positive ion fast atom bombardment mass spectrometry for rapid identification of a bile alcohol glucuronide isolated from cerebrotendinous xanthomatosis patients

The identification of a major biliary and plasma bile alcohol glucuronide, 5β-cholestane-3α,7α,12α,25-tetrol-3-O-β- d-glucuronide, present in cerebrotendinous xanthomatosis (CTX) patients, was investigated by positive ion fast atom bombardment mass spectrometry (FAB-MS). The spectrum was characterised by abundant ions formed by attachment of a proton, [M + H] +, or of alkali ions, [M + Na] + and [M + 39K] +, to the glucuronide salt. These ions allowed an unambiguous deduction of the molecular weight of the sample. It is suggested that FAB-MS could be used m the rapid diagnosis of CTX. (Steroids 55:74–78, 1990)

N-Formyl-2-aminoethanol as a matrix for fast atom bombardment mass spectrometry of non-polar biomolecules

N-Formyl-2-aminoethanol appears to be a very suitable matrix for the fast atom bombardment (FAB) mass spectrometry of many (bio)organic molecules. The best results so far have been obtained with compounds having a decreased hydrophilic character. These include (per)methylated oligosaccharides and peptides containing relatively many amino acids with non-polar side-chains. The positive and negative ion FAB mass spectra of these compounds show abundant [M + H] +, [M − H] − and sequence ions, while the ions resulting from the matrix do not dominate.

FAB-MS/MS spectrometry in determining the primary structure of .GAMMA.-glutamyl-containing peptides.

Positive fast atom bombardment tandem mass spectrometry (FAB-MS/MS) was applied for peptide sequencing, particularly for determining the gamma glutamyl linkage involved in metal-binding peptides such as Cadystin (gamma EC)3G = Cadystin A and Cadystin (gamma EC)2G = Cadystin B (MW 771 and 539, respectively). The fragmentation patterns between the natural gamma glutamyl peptide and the synthetic alpha glutamyl one were clearly distinguishable. FAB-MS/MS was proved to be a good method for determining these peptides, since it needed no chemical degradation and only a small amount of the peptide was needed for determination.

Methodological approach to the characterization of diacylphosphatidyl cholines in rabbit lung lavage fluid by fast‐atom bombardment mass spectrometry

Strong evidence exists that the distribution of phospholipids in the lung is a function of the degree of adult respiratory distress syndrome. The capabilities of positive-ion fast-atom bombardment (FAB) mass spectrometry for the determination of the relative distribution of intact diacylphosphatidylcholine species in lung lavage fluid were investigated. Two different FAB matrices and two different isolation/purification procedures--extraction/thin layer chromatography--high performance liquid chromatography (TLC-HPLC)--have been evaluated. In addition the relative fatty acid composition of the diacylphosphatidylcholines was determined by negative-ion FAB mass spectrometry using the [RCOO]- fragment ion. These results were compared with those obtained by gas chromatographic determination of the fatty acid methylesters.

Analysis of glycolipids by fast atom bombardment mass spectrometry

The positive and negative ion fast atom bombardment (FAB) mass spectra of four glycolipids obtained from microbial cultures are reported. The spectra of the glycolipids in the positive ion mode are characterized by abundant [M + Na]+, [M + Na + matrix]+ and [M + 2Na - H]+ species. In negative FAB conditions the molecules yield [M - H]-. Our understanding of the FAB behaviour of glycolipids in both positive and negative modes has been considerably aided in the structure elucidation, without any derivatization or degradation reaction of the compounds studied. The technique allows unambiguous molecular weight determination of low-microgram amounts of these glycolipids purified from biological sources and provides useful fragmentation information.

The N-linked oligosaccharides of bovine skin proteodermatan sulphate.

activity was collected and rotary evaporated to dryness. The residue was extracted ( x 5) with 10 ml of methanol, redissolving the dried residue in water and drying before each extraction. The pooled methanol extracts were dried under vacuum, redissolved in 1 ml of methanol/chloroform/2.5 Mammonium hydroxide (40:60:8; by vol.) and run onto a silica gel column (50 cm x 1 cm) equilibrated with the same solvent at 0.5 ml/min. Fractions (10 ml) were collected and inhibitor was shown t o be eluted between fractions 3 and 1 0 . Fractions 4-7 inclusivc were pooled, rotary evaporated to dryness then redissolved in ammonium bicarbonate (10 mM). The sample was finally purified by h.p.1.c. using a C , , (Spherisorb ODS. 25 cm x 4.6 mm) column equilibrated in 10 mM-ammonium bicarbonate and run at 1.5 ml/min. After loading of the sample, a linear gradient from 10% to 50% methanol was applied over a 25-min period. The inhibitor eluted between I 1 and 13 min and gave a single spot ( H , = 0.22) t.1.c. silica plates in methanol/chloroform/2.5 M-ammonium bicarbonate (40:60:8, by vol.). when developed with 50% (v/v) sulphuric acid. Development with phenol sulphuric acid gave a brown spot and carbazole sulphuric acid gave a violet spot, indicating the presence of a sugar. Positive fast atom bombardment mass spectroscopy on the purified material gave an M , of 323. Further study using chemical ionization of the trimethylsilylated derivative and analysis by nuclear magnetic resonance has provided the proposed structure shown in Fig. 1 . Methanolic extracts of the algae Mesotueriiitm culduriorum and Mougeotiu sp. have been shown to inhibit a-glucosidase. For both extracts the active components have been extensively purified by chromatography on Sephadex LH 2 0 (41 c m x 1.6 cm) in 95% (v/v) methanol/S% (w/v) ammonium bicarbonate/100 mM-&mercaptoethanol, followed by chromatography on silica in water containing 100 mM-~-mcrcaptocthanol for Mesotueriiitm culdurioriim and 80%) (v /v) water/20% (v/v) methanol/ 100 mM-pmercaptoethanol for Moitgeotiu sp. The active fractions were collected and each showed a single spot on silica t.1.c. (butanol/acetic acid/water, 2.8: 1 : 1, by vol.) when developed with SO‘% (v/v) sulphuric acid. However, in both cases, attempts at further analysis by h.p.1.c. and mass spectroscopy were unsuccessful due to the consistent loss of activity on concentration or drying of the sample. Concurrent with the loss of activity was the appearance of a reddish brown precipitate in each sample. Such changes are symptomatic of the polymerization o f tannins, which can be caused by air oxidation [ 41, a not uncommon problem associated with the purification o f tannins. Use of excess P-mercaptoethanol did not

Bis(tertiary amine)dihaloboron cations and related species: nuclear magnetic resonance and fast atom bombardment mass spectrometry studies

The formation of four-coordinate haloboron cations from aliphatic tertiary amine adducts of the mixed boron trihalides by heavy halogen displacement has been systematically studied by 19F and 11B nuclear magnetic resonance and positive ion fast atom bombardment mass spectrometry (FAB). Low-steric-hindrance donor molecules readily displace bromide ion from tertiary amine – bromodifluoroborane adducts, D•BF2Br, to form difluoroboron cations D2BF2+ and DD′BF2+, but the corresponding dibromofiuoroborane and tribromoborane adducts are highly resistant to bromide ion displacement. Bis(tertiary amine)dichloroboron and -chloroiodoboron cations can be obtained by selective iodide displacement from D•BCl2I and D•BClI2. Fast atom bombardment mass spectrometry selectively detects the haloboron cations in preference to the neutral adducts in mixtures, and is a valuable complement to nmr in monitoring formation of the haloboron cations as well as any ionic by-products. Keywords: difluoroboron cations, dihaloboron cations, NMR, 11B, 19F, fast atom bombardment (FAB), ligand substitution, redistribution reactions.