New approach for characterization of lysosulfatide by TLC, fast atom bombardment mass spectrometry and NMR spectroscopy

Thin layer chromatography of lysosulfatide showed anomalous Rf-values in contrast with such lysosphingolipids as glucopsychosine and galactopsychosine with neutral, acidic, and alkaline developing solvents. This was thought to be due to the presence of oppositely charged sulfate and amino groups in the lysosulfatide. In the negative mode of fast atom bombardment mass spectrometry, the lysosulfatide showed the pseudo molecular ion (M-H)- peak at m/z 540 and sulfate ion peak at m/z 97, whereas in the positive mode, it showed not only the pseudo molecular ion (M+H)+ peak at m/z 542, but also the major peaks of protonated psychosine at m/z 462 and fragment ions of dehydrated sphingosine at m/z 282 and 264, 13C-NMR signals of all carbons of lysosulfatide were determined by using distortionless enhancement by polarization transfer. The difference in chemical shifts of ring carbons of galactose residue between lysosulfatide and galactopsychosine was largest at C-3 (downfield shift), thereby indicating the location of the sulfate group to be at C-3 of galactose. This conclusion is supported by the 1H-NMR spectra of the lysosulfatide and galactopsychosine. Thus, the chemical structure of lysosulfatide was confirmed by fast atom bombardment mass spectrometry and 13C- and 1H-NMR spectroscopy. Furthermore, 13C-NMR signals of C-1 to C-5 of the sphingosine moiety showed significantly different chemical shifts between the lysosulfatide and galactopsychosine. These differences suggested that C-1 to C-5 of sphingosine might be influenced by intramolecular or intermolecular interaction between the sulfate group of the galactose residue and the amino group of sphingosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide studies using a fast atom bombardment high field mass spectrometer and data system. 3--Negative ionization: mass calibration, data acquisition and structural characterization

Under negative ionization conditions, nominal mass calibration of the fast bombardment high field mass spectrometer and data system was accomplished using cesium iodide/glycerol as a reference. Mass calibration at --8 kV accelerating potential extends from m/z 387 to m/z 2170 using xenon fast atoms. Negative xenon FAB mass spectra for human angiotensin I and human gastrin I complement their positive fast atom bombardment spectra. Negative xenon fast atom bombardment spectra of underivatized peptides exhibit molecular proton-abstracted ion envelopes and structurally significant fragment ions. Peptide mixture analysis under negative xenon fast atom bombardment reveals peptide molecular ion envelopes of higher relative intensities than under positive xenon fast atom bombardment.     

Fast ATOM Bombardment Mass Spectra of Nucleosides. Comparison with Electron Impact and Chemical Ionization Mass Spectra

Abstract

The fast atom bombardment (FAB) mass spectra of the eight major nucleosides found in RNA and DNA, pseudouridine and 2′,3′-O-isopropylidene adenosine are described and compared to El, CI, and desorption chemical ionization (DCI) spectra reported in the literature or obtained in this laboratory. Bcty, cocltl nun FAB spectra are reported. The FAB spectra are simple and provide unambiguous molecular weight information along with structurally significant fragment ions. Mechanisms of ion formation are thought to closely parallel those suggested earlier to be operating in the CI mode. Advantages and disadvantages of FAB relative to the standard ionization modes are discussed.     

Fast atom bombardment mass spectrometry and tandem mass spectrometry in antibiotics: identification of nucleoside antitumor antibiotic toyocamycin in fermentation broth

The presence of the nucleoside antitumor antibiotic toyocamycin in the fermentation broth was determined by a combination of negative and positive ion fast atom bombardment (FAB) mass spectrometry, high resolution FAB mass spectrometry and mass-analysed ion kinetic energy spectrometry (MIKES). A reasonable limit of detection for toyocamycin in the whole broth was obtained by combining the specificity of mass spectrometry/mass spectrometry (also called tandem mass spectrometry) to FAB. The role played by the fermentation matrix upon the production and the observation of characteristic ions by FAB using xenon atoms was examined. High-performance liquid chromatography (HPLC) and FAB mass spectrometry were used to monitor toyocamycin at all stages of strain development, fermentation and recovery.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

The electron impact, chemical ionization and fast atom bombardment positive ion mass spectra of 1,2-bis(sulfonyl)methylhydrazines.

A series of bis(sulfonyl)-1-methylhydrazines were analyzed by positive ion electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry. Since these compounds showed activity against the L1210 leukemia, an understanding of their mass spectral behavior is important should the structural characterization of metabolites be required. FAB proved to be the most useful technique, generally providing abundant protonated molecule ion peaks, in contrast to the weak peaks observed with CI (ammonia or isobutane) and the total absence of molecular ion peaks in the EI mass spectra. In addition, utilizing FAB eliminated the problem of thermal decomposition, which was very difficult to control under EI and CI experimental conditions. Fragments observed in FAB and CI mass spectra were consistent with protonation at the methyl-bearing nitrogen. One can locate the R1 and R2 moieties relative to the methyl-bearing nitrogen in FAB and CI by assigning that nitrogen as the site of protonation, with subsequent elimination of R2SO2H.     

Detection of D-erythro and L-threo sphingosine bases in preparative sphingosylphosphorylcholine and its N-acylated derivatives and some evidence of their different chemical configurations

Sphingosylphosphorylcholine prepared from native sphingomyelin by the Kaller procedure was found to comprise about 70% of the L-threo (2S, 3S) isomer and 30% of the D-erythro (2S, 3R) isomer. This analytical result was obtained by gas-liquid chromatography (GLC) of trimethylsilyl derivatives of N-acetylsphingosines which were prepared by enzymatic hydrolysis of synthetic N-acetylsphingosylphosphorylcholines with Clostridium perfringens phospholipase C. Some other evidence of the different chemical configuration between the erythro and threo isomers of synthetic N-acylated sphingosylphosphorylcholines was also provided by thin layer chromatography (TLC), optical rotatory dispersion (ORD), and fast atom bombardment (FAB) mass spectrometry.     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

Structural studies of gangliosides by fast atom bombardment ionization, low-energy collision-activated dissociation, and tandem mass spectrometry

Negative ion fast atom bombardment, low-energy collision-activated dissociation, and tandem mass spectrometry techniques were applied for the structural elucidation of gangliosides. The mass spectra were simplified by selecting a single molecular ion or fragment ion in the analysis of mixtures, and interference by background signals from the liquid matrix could be avoided. Introduction of collision-activated dissociation produced abundant fragment ions convenient for structural analysis. In the daughter scan mode, ions were produced by cleavage of the glycosidic bonds, and not by cleavage at the sugar ring. These ions all contain ceramide moieties, except the sialic acid fragment ion. In the parent scan mode, product ions resulting from cleavage at the sugar ring were detected beside the ions resulting from cleavage at the glycosidic bonds, and ions of oligosaccharide fragments were also detected. In parent scan mode spectra of gangliosides based on the sialic acid ion, all ions contained a sialic acid residue, and the observed ions were similar to those obtained in the high-energy collision-activated dissociation daughter scan mode. These results indicate the usefulness of low-energy collision-activated dissociation tandem mass spectrometry in the daughter and parent scan modes for the analysis of ganglioside structure, in combination with fast atom bombardment mass spectrometry and high-energy collision-activated dissociation mass spectrometry.     

Characterization of lipid A from Pseudomonas aeruginosa O-antigenic B band lipopolysaccharide by 1D and 2D NMR and mass spectral analysis.

The Lipid A from the lipopolysaccharide of Pseudomonas aeruginosa was examined by high-field nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The backbone structure and the position of phosphate substituents were unambiguously established by one- and two-dimensional 1H, 13C, and 31P NMR techniques on a de-O-acylated Lipid A sample. The Lipid A has a beta-(1,6)-glucosamine disaccharide structure which is substituted by phosphomonoesters through glycosidic bonds at C-1 and at C-4'. The configuration of the glycosidically linked phosphate at position C-1 was identified as alpha which is the same as that of Enterobacterial Lipid A. Chemical analysis revealed that the Lipid A contained 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, 3-hydroxydecanoic, and hexadecanoic acids in the approximate molar ratios 2.2:2.0:0.2:0.8:0.4. From 1H NMR and fast atom bombardment (FAB) mass spectrometry on the de-O-acylated Lipid A, it was established that both glucosamine residues were N-acylated by 3-hydroxydodecanoic acid. The identity and positions of the ester bound fatty acids in the intact Lipid A were investigated by negative ion FAB-MS. In addition to the hexaacyl and pentaacyl Lipid A species, a tetraacyl species was identified. Heterogeneity due to hydroxylated and nonhydroxylated dodecanoic acid esters could be uniquely localized to the nonreducing beta-glucosamine residue from the fragmentation pattern observed in the negative ion FAB-MS. The complete structure of the Lipid A from P. aeruginosa will be useful in understanding the determinants responsible for the endotoxin activity of this molecule.     

Fast atom bombardment mass spectrometry of estrogen glucuronides and sulfates

Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are characterized by abundant ions formed by attachment of a proton, [M+H]+, or of an alkali ion, [M+alkali]+, to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to obtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt.     

Comparison of 252californium plasma desorption and fast atom bombardment mass spectrometry for analysis of small peptides

The data obtained with 252Cf plasma desorption (PD) and fast atom bombardment mass spectrometry of eight tri-, tetra- and pentapeptides were compared. Good spectra were obtained with 1-10 nmol of peptide. In both techniques molecular weight information was obtained. The PD mass spectra are often dominated by the cationized molecular ions in contrast to the fast atom bombardment (FAB) mass spectra, where cationization is rarely observed. Amino acid content is reflected in the immonium ions equally well in both techniques. The fragmentation patterns observed with the two techniques are almost identical. However, practical sequencing of peptides based on either FAB or PD mass spectrometry of underivatized peptides alone is difficult. This is due to the unpredictable and sometimes absent cleavage yield at certain peptide bonds. Another difficulty is the many simultaneous fragmentation pathways. However, for many peptides enough information is present to allow sequence determination for at least a major part of the molecule.     

Identification and quantification of metabolite conjugates of activated cyclophosphamide and ifosfamide with mesna in urine by ion-pair extraction and fast atom bombardment mass spectrometry

The high bladder toxicity of the alkylating oxazaphosphorine anticancer drugs, cyclophosphamide and ifosfamide is effectively reduced by the concomitant administration of mesna (sodium 2-mercaptoethane sulphonate). The formation and rapid urinary excretion of conjugates of the activated (4-hydroxylated) oxazaphosphorine metabolites with mesna has been suggested as the pharmacological basis for the selective detoxification, but separation and identification of such metabolites in vivo have been extremely difficult due to their high polarity and chemical lability. In this study an ion-pair extraction procedure in combination with positive and negative ion fast atom bombardment mass spectrometry has been developed which enabled the identification and quantification of the conjugation products of activated oxazaphosphorine metabolites with mesna in urine. The conjugates extracted as the tetra-n-butylammonium salts are directly identified by their characteristic positive molecular ion adducts and fragment ions, and the corresponding abundant molecular anions. The pattern of molecular and fragment ion formation was established by comparison of the fast atom bombardment mass spectra of synthetic cyclophosphamide-mesna conjugates with various organic and inorganic counter ions. The ifosfamide-4-(2-thioethylsulphonate) (ifosfamide-mesna) conjugate was identified as a metabolite in the urine of rats, and in patients after administration of the combination, ifosfamide + mesna. By means of a two-step extraction and with the use of suitable analogues as internal standards, procedures for the quantification of parent oxazaphosphorine and of oxazaphosphorine-mesna conjugates by negative ion fast atom bombardment mass spectrometry have been developed, and first examples for the determination of excretion kinetics are described.     

The synthesis and characterization of nucleotide complexes involving an asymmetric metal

The synthesis of cobalt and chromium complexes of H₄ATP and H₄GTP in which the metal is asymmetric are reported. These compounds were characterized by visible spectroscopy, fast atom bombardment mass spectroscopy (FAB MS), and ³¹P NMR. The mass spectral data allow identification of the complexes to be made from ions in the molecular weight region. The effect of an asymmetric metal greatly alters the appearance of the ³¹P NMR spectra in comparison to complexes which do not have this feature. Complexes of uridine diphosphoglucose, UDPG, are also reported. The effect of an asymmetric metal ion on the chromatographic and spectral properties of the complexes are discussed.     

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin

Summary A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by¹H,¹H-¹H,¹³C and¹H-¹³C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.     

A unique fucoganglioside with blood group B determinant in rat spleen

A unique fucoganglioside was isolated from rat spleen and characterized by compositional analysis, methylation analysis, exoglycosidase treatment, negative ion fast atom bombardment (FAB) mass spectrometry, and proton NMR spectrometry. The ganglioside was identified as alpha Gal,Fuc-GM1(NeuGc), which has the blood group B determinant at the nonreducing termini, as shown below: (formula; see text) This is the first report describing the occurrence in nature of alpha Gal,Fuc-GM1 containing N-glycolylneuraminic acid.     

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.     

Fast atom bombardment tandem mass spectrometric analysis of N-carbamoylamino acids.

Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.     

Fast atom bombardment and collisional activation mass spectrometry of retinyl phosphate mannose synthesized by liver membranes

Fast atom bombardment (FAB) and collisional activation dissociation (CAD) mass-analysed ion kinetic energy (MIKE) spectra have confirmed the structures of retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and guanosine 5'-diphospho-D-mannose (GDP-Man). Ret-P-Man was made in vitro while Ret-P and GDP-Man were chemically synthesized. Positive ion FAB mass spectrometry of Ret-P showed an observable short-lived spectrum with a mass ion at m/z 367 [M + H]+, and a major fragment ion at m/z 269 [M + H - H3PO4]+. Negative ion FAB mass spectrometry of Ret-P showed a strong stable spectrum with a parent ion at m/z 365 [M - H]-, a glycerol (G) adduct ion at m/z 457 [M - H + G]- and a dimer ion at m/z 731 [2M - H]-. GDP-Man showed an intense spectrum with parent ion at m/z 604 [M - H]- and cationized species at m/z 626 [M + Na - 2H]- and 648 [M + 2Na - 3H]-. Negative ion FAB mass spectrometry of Ret-P-Man showed a parent ion at m/z 527 [M - H]- and a fragment ion at m/z 259 [C6H12PO9]-. The CAD-MIKE spectra showed structurally significant fragment ions at m/z 442 and 361 for the [M - H]- ion of GDP-Man, and at m/z 509, 406, 364 and 241 for the [M - H]- ion of Ret-P-Man. FAB and CAD-MIKE spectra have been applied successfully to confirm the structure of Ret-P-Man made in vitro from Ret-P and GDP-Man.     

A novel mass spectrometric procedure for the rapid determination of the types of carbohydrate chains present in glycoproteins: application to alpha-galactosidase I from Vicia faba seeds

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains.