Positive-ion Fast-atom Bombardment Mass Spectrometry Research Articles

Kinetic analysis of cyclic CMP-specific and multifunctional phosphodiesterases by quantitative positive-ion fast-atom bombardment mass spectrometry.

Two enzymes, cyclic CMP-specific phosphodiesterase and multifunctional phosphodiesterase, are responsible for the hydrolysis of cytidine 3',5'-cyclic monophosphate in living cells. Quantitation of both enzymes has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubates after termination of the reaction. The kinetic data obtained are in close agreement with parallel data obtained by the conventional radiometric assay. The extra facility of the mass spectrometry based assay to monitor several incubation components simultaneously has been exploited to study the concurrent hydrolysis of alternate cyclic nucleotide substrates and provides kinetic parameters of significance in interpreting substrate-enzyme interactions. This is extended by the use of collisionally-induced dissociation of the protonated molecules of the liberated products to identify the mononucleotide isomers resulting from the cyclic nucleotide hydrolysis.

Differences in the Formation and Fragmentation of Sodium Adduct Ions between Tertiarybutoxycarbonyl-Protected Prolylproline Diastereomers in Fast Atom Bombardment Mass Spectrometry.

The effect of Na+ ions on the fragmentation of tertiarybutoxycarbonyl (Boc) protected prolylproline (Pro-Pro) diastereomers, Boc-Pro-Pro and Boc-D-Pro-Pro, was studied in positive-ion fast atom bombardment (FAB) and tandem mass spectrometry. The formaiton of the [M+Na]+ ion for Boc-D-Pro-Pro was more predominant than that for Boc-Pro-Pro on the addition of sodium chloride in positive-ion FAB mass spectrometry, suggesting that Boc-D-Pro-Pro has a stronger Na+ ion affinity than Boc-Pro-Pro. In the collisional-activated decomposition mass spectra of the [M+Na]+ ions, the abundance of the [M+Na-C(CH3)3+H]+ ion, which is due to the loss of a tertiarybutyl group from the [M+Na]+ ion for Boc-D-Pro-Pro, was higher than that for Bos-Pro-Pro. These results indicate that the interaction of the Na+ ion with Boc-Pro-Pro is different from that with Boc-D-Pro-Pro in the FAB condition, and these diastereomers are distinguished by the addition of a Na+ ion in FAB and tandem mass spectrometry.

Disulfated oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis

Twenty novel disulfated oligosaccharides were purified in nanomolar quantities from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, followed by anion-exchange chromatography, size-exclusion chromatography, and high-performance liquid chromatography (HPLC). In addition to positive ion fast-atom-bombardment mass spectrometry (FABMS), proposed structures for the resulting purified disulfated oligosaccharides were also based on carbohydrate permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, and parallel analysis of desulfated products. Sulfate esters were found to reside on C-3 or C-6 of terminal d-galactose and on C-6 of internal d-galactose or 2-acetamido-2-deoxy- d-glucose residues. For this group of oligosaccharides, ranging in size from tri- to undeca-saccharides and possessing linear, di- and tri-antennary forms, it was also observed that sulfate esters could be located on the same or on different branches and that branched oligosaccharides can possess sulfate esters on C-3 and C-6 of different terminal galactose residues within the same structure.

Preparation of Cu(II) complexes of oxidized chlorophylls and their determination by thin-layer and high-performance liquid chromatography

Abstract Different allomerization products of pheophytins a and b have been obtained by alkaline treatment in the presence of atmospheric oxygen and isolated by normal phase thin-layer chromatography (NP-TLC) and semi-preparative high-performance liquid chromatography (HPLC). 3 1 ,3 2 -Didehydro-151-hydroxy-15 1 -hydroxy-rhodochlorin-15-acetic acid δ-lactone-15 2 -methyl-17 3 -phytyl ester ( a and b ) and 3 1 ,3 2 -Didehydro-rhodochlorin-15-glyoxylic acid-17 3 -phytyl ester ( a and b ) have been identified as principal products. Identification was based on UV-Vis and mass spectra, retention times and R F values. The molecular mass of allomerized pheophytins was confirmed by positive-ion fast-atom bombardment mass spectrometry and positive/negative ion electrospray mass spectrometry. The corresponding Cu(II) complexes of these compounds were prepared by chelation reaction with CuCl 2 and separated by reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC) using a gradient of water and ion suppressor-methanol-acetone. The Chromatographic characteristics in NP-TLC and RP-IP-HPLC of the Cu(II) complexes are reported. The corresponding UV-Vis spectra obtained with an on-line diode-array detector have been included.

Positive-ion fast-atom bombardment mass spectrometry of nitrogen macrocycles

Fast-atom bombardment (FAB) mass Spectra of nitrogen macrocycles studied in this work show fragmentations which occur by loss of neutral molecules from the protonated molelcule to give even-electron ions. Furthermore, both the neutral molecules and the even-electron ions characterize substructures of each compound studied. Thus, it is easy to identify an unknown nitrogen macrocycle from its FAB mass Spectrum.

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.

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.

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Copper has been reported to promote the demethylation of N-methylporphyrins. Copper-induced dealkylation is observed at room temperature in acetonitrile in the presence of a suitable nucleophilic acceptor molecule, or in chloroform alone. Electronic absorption spectra of N-methylprotoporphyrin IX (in the form of its dimethyl ester) in the presence of copper (with either chloroform alone or acetonitrile-dibutylamine as solvent) exhibited a transient spectrum corresponding to a copper chelate of the N-methylprotoporphyrin followed by ejection of the N-methyl group to afford the copper chelate of protoporphyrin. The use of fast atom bombardment mass spectrometry (FAB-MS) to identify the N-alkyl group of a series of N-substituted porphyrins was investigated. The alkyl group released on reaction with copper ions was trapped by an amine (e.g., dodecylamine). The resulting mixture was analysed by positive-ion FAB-MS using 3-nitrobenzyl alcohol as matrix. Molecular ions (MH +) were detected at m/z 200, 214 and 228, corresponding to [CH 3(CH 2) 11NHCH 3 + H] +, [CH 3(CH 2) 11NHC 2H 5 + H] + and [CH 3(CH 2) 11NHC 3H 7 + H] +, respectively. The analysis was extended to N-alkylprotoporphyrins isolated from liver homogenates of mice treated with the porphyria-inducing drug griseofulvin. Using a combination of UV and copper-induced dealkylation studies in FAB-MS, the porphyria-producing pigment was identified as N-methylprotoporphyrin IX.

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.