Urine from patients with calcium-4-(2,4-dihydroxy-3,3-dimethylbutyramido) butyrate hemihydrate (hopantenate) therapy during episodes of Reye's-like syndrome was found to contain a number of unusual dicarboxylic acids in high concentrations; odd- and even-numbered medium-chain dicarboxylic acids, α-hydroxydicarboxylic acids and β-hydroxydicarboxylic acids. The abnormal excretion of dicarboxylic acids, α- and β-hydroxydicarboxylic acids disappeared after discontinuance of hopantenate therapy. Besides the excretion of 2-hydroxydecandedioic acid, which has been previously described in Zellweger syndrome or neonatal adrenoleukodystrophy, a series of α-hydroxydicarboxylic acids was detected and identified. In this paper, we have characterized some new compounds by gas chromatography/mass spectrometry: 2-hydroxydodecanedioic acid, 2-hydroxydodecenedioic acid, 2-hydroxytetradecanedioic acid, 2-hydroxytetradecenedioic acid and 2-hydroxyoctanedioic acid.
Collision-induced dissociation of protonated molecular ions in a mixture of macrotetralide antibiotics has been used to determine the sample make-up, as well as the structures of the components in the mixture. Upon 3 keV gaseous collisional activation the near-tetrameric structure of the macrotetralides is opened and can then fragment further to give oligomeric ions through consecutive losses of monomeric units. The fragment ions generated indicate which monomers are present and also their sequence in the cyclic assembly. The fragmentation mechanism involves cleavage of an ester bond to give an undissociated carboxyl group. This cleavage may take place remote from the charge site.
The use of chloroacetonitrile as eluent additive in thermospray liquid chromatography/mass spectrometry in the negative ion mode using filament-off, filament-on and discharge ionization modes has been studied with chlorophenols and chlorinated phenoxy acids as test compounds. In the absence of chloroacetonitrile from the eluent, different base peaks were observed corresponding to [ M H]−, [ M + H]− and [ M + CH3COOO]−, while in the presence of 2% chloroacetonitrile [M + Cl] − was the base peak in most cases when using the filament-on and discharge ionization modes and had a maximum value of 30% relative abundance in the filament-off mode. Most chloride attachment ([ M + Cl] − ion) was always observed in the filament-on mode. Other ions observed were adduct ions with the ionizing additive (ammonium acetate) such as [M + CH3COO]− and [ M + (CH3COOH). (CH3COO)]− when the eluent contained no chloroacetonitrile and [ M + (CH3COOH). Cl]− when 2% chloroacetonitrile was present. The use of chloroacetonitrile in the eluent provides additional structural information in thermospray liquid chromatography/negative ion mass spectrometry. It does not cause any appreciable loss in sensitivity during flow injection or chromatographic analysis.
A rotatable dual-target probe was used for accurate mass measurement in fast atom bombardment mass spectrometry to determine the structures of unknown amino acid residues or post-translationally modified structures in peptides or proteins. The results obtained in measurement of tryptic peptides (with molecular weights of up to 2000) of the A-subunit of vero-toxin I indicated that the mass values obtained are sufficiently accurate and reproducible to allow the generation of the possible elemental compositions for modifications in peptides. By this method, the structural modifications of the N-terminal of a recombinant human leukocyte interferon A and novel halogenated amino acids in sperm-activating peptides from the egg-jelly of sea urchins were determined.
A quantitative and selective method for analysing indole alkaloids from complex mixtures was established by combining high-performance liquid chromatography with mass spectrometry using a thermospray sample-feeding principle. Detection limit was 100 pg per injection and response was linear up to 200 ng for all the compounds (catharanthine, tabersonine, serpentine and ajmalicine) that were to be analysed. The compounds were quite stable under conditions used, and the protonated molecular ions appeared as base peaks in the mass spectra and were selected for ion recording. An application of the assay for monitoring contents of the alkaloids in cell cultures derived from Catharanthus roseus is described in detail.
With the aid of a new chiral derivatizing reagent and a sensitive and specific assay using capillary gas chromatography/negative ion chemical ionization mass spectrometry, the proportions of R and S enantiomers of 1-methyl-1, 2, 3, 4-tetrahydroisoquinoline (1MeTIQ) in mammalian tissues and foods were studied. R- and S-1MeTIQ enantiomers derivatized with a chiral derivatizing reagent, perfluoro-2-propoxypropionylchloride, were clearly separated. The ratio of enantiomers was R/S = 0.24, 0.55 and 0.60 in wine, cocoa and mouse brain. S-1MeTIQ predominated in all samples. This result suggested that 1MeTIQ could be formed at least partially through an enzymatic mechanism.
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.
Eight metabolites of retinol were isolated by high-performance liquid chromatography (HPLC) from the plasma of the non-human primate Macaca fascicularis after acute exposure to 150,000 IU of vitamin A per kilogram body weight. After enrichment and further chromatographic purification, the metabolites were reinjected individually into a second HPLC system which was connected on-line by a thermospray interface to a mass spectrometer operated in the positive ionization mode. Six retinoids were identified by (i) a comparison of their retention times with those of appropriate reference compounds in the two chromatographic systems and (ii) by comparison of their mass spectra with those of reference compounds. These retinoids were: 13-cis-4-oxoretinoic acid, all-trans-4-oxoretinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, all-trans-retinoyl beta-glucuronide and all-trans-retinyl beta-glucuronide. One further metabolite could be identified for the first time as all-trans-4-oxoretinoyl beta-glucuronide by its mass spectrum and, after treatment of the unknown metabolite with beta-glucuronidase, by its hydrolysis product all-trans-4-oxoretinoic acid. The molecular structure of one metabolite could not be elucidated. A major metabolic pathway of high-dose vitamin A in the non-human primate is apparently the oxidation of the primary alcohol group of retinol resulting in the formation of all-trans-retinoic acid. Subsequently, a broad spectrum of various metabolites of all-trans-retinoic acid, including beta-glucuronides and retinoids with a 13-cis configuration, appear in the plasma.
A new procedure using fast atom bombardment mass spectrometry for the determination of arginine residues in neuropeptides is described. The technique is based on the modification of the arginine side-group with 1,2-cyclohexanedione. This novel procedure may have a potential use in the study of neuropeptides and their processing and degrading enzymes.
Thermospray mass spectrometry of inositol mono- and polyphosphates, separated by ion-exchange chromatography, was evaluated for its potential as a general method for quantitative analysis of these substances. The only ions of significant abundance that are produced by the thermospray ionization process result from the total loss of phosphate from inositol. Thus inositol mono-, tris- and hexakisphosphate each gave mass spectra consisting solely of [MH]+ and [MNH4]+ of inositol. When the chromatographic eluates are passed through a heated reactor prior to the thermospray source maximal yields of these ions are obtained. The sensitivity of the technique falls short of that needed for a general method for biological applications, because the lower limit of detection is about 100 pmol μl−1. Inositol phosphates peracetylated on C-hydroxyls were also studied, with separation by ion-exchange chromatography. Again, thermospray ionization produces totally dephosphorylated species, with the highest-mass ions retaining all of the acetyl groups, even when using the thermal reactor. Losses of acetate were also observed. Sensitivity with the acetyl derivative was comparable to that with the underivatized inositol phosphates.
Schistosomiasis is a parasitic liver infection which is known to affect many aspects of drug metabolism. Praziquantel (PZQ) is the drug of choice for treating this disease. PZQ is known to be highly metabolized, but the effect of the disease on its metabolism has not been investigated. Control mice and mice infected with Schistosoma mansoni were dosed with PZQ and their urines were examined for the presence of metabolites using a triple-quadrupole mass spectrometer (tandem mass spectrometer). The collisionally induced dissociation of PZQ was remarkable in its structurally significant fragments. From this we were able to identify 17 hydroxylated metabolites of PZQ from purified urine samples without further chemical separation, including three monohydroxylated, six dihydroxylated, and eight trihydroxylated metabolites. There were no qualitative differences in metabolite production between control and infected animals.
Biomedical & Environmental Mass SpectrometryVolume 19, Issue 6 p. 390-392 Short Communication High performance tandem mass spectrometry for sequence, branching and interglycosidic linkage analysis of peracetylated oligosaccharides B. Domon, B. Domon Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorD. R. Müller, D. R. Müller Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorW. J. Richter, W. J. Richter Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this author B. Domon, B. Domon Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorD. R. Müller, D. R. Müller Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorW. J. Richter, W. J. Richter Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this author First published: June 1990 https://doi.org/10.1002/bms.1200190611Citations: 46AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 B. Lindberg and J. Lönngren, in Methods in Enzymology ed. by V. Ginsburg, Vol. 50, p. 3, Academic Press, New York (1978). 2 H. Egge and J. Peter-Katalinic, Mass Spectrom. Rev. 6, 331 (1987). 3 S. A. Carr, V. N. Reinhold, B. N. Green and J. R. Hass, Biomed. Mass Spectrom. 12, 288 (1985). 4 S. Santikarn, V. N. Reinhold, C. E. Costello and C. Warren, Proc. 35th Conf. Mass Spectrom. Allied Topics, Denver, CO, p. 870 (1987). 5 B. L. Gillece-Castro and A. L. Burlingame, Proc. 35th Conf. Mass Spectrom. Allied Topics, Miami Beach, FL, p. 1190 (1989). 6 B. Domon, D. R. Müller and W. J. Richter, Org. Mass Spectrom. 24, 357 (1989). 7 D. R. Müller, B. Domon and W. J. Richter, in Advances Mass Spectrometry, ed. by P. Longevialle, Vol. 11B, p. 1309, Heyden & Son Ltd, London (1989). 8 D. R. Müller, B. Domon and W. J. Richter, in Methods in Enzymology, ed. by J. A. McCloskey, Academic Press, Orlando FL (1990), in press. 9 B. Domon, D. R. Müller and W. J. Richter, submitted for publication. 10 B. Domon and C. E. Costello, Glycoconj. J. 5, 397 (1988). Citing Literature Volume19, Issue6June 1990Pages 390-392 ReferencesRelatedInformation
Two novel gas chromatography/mass spectrometry (GC/MS) strategies have been developed to assist in characterization of ganglioside inner esters. In the first, lactonized gangliosides which have been ammonolyzed and permethylated (see previous paper) are subjected to methanolysis and acetylation. This produces partially methylated NeuAc derivatives in which previous involvement of the carboxyl group in an ester linkage is indicated by conversion to an N1, N1-dimethylamide. These can be analyzed by GC/MS using conditions analogous to those used normally in NeuAc methylation analysis, providing glycosidic and ester linkage information in the same procedure. In the second procedure, internally esterified NeuAc hydroxyl groups are located by protection, under neutral conditions, of all free hydroxyl groups of the ganglioside lactone with methoxyethoxymethyl groups, followed by permethylation, methanolysis, and GC/MS analysis of the resulting partially methylated NeuAc methyl ester methyl glycosides as their per-O-trimethylsilylates or per-O-acetates. This procedure has been used to show unambiguously that in GD3 lactones it is the 9-hydroxyl group of the internal NeuAc residue which is esterified by the terminal NeuAc carboxyl. Both of these strategies should have general application in characterizing lactones occurring in gangliosides, NeuAc homo- and heteropolymers, and oligosaccharides or glycopeptides terminated by polysialosyl chains.
The relative response of various perfluorocarbons (PFCs) such as perfluorinated alkanes, alkenes, aromatics and heteroatom-containing fluorocarbons such as perfluorinated 2-butyltetrahydrofuran, tert-butanol, bis(isopropyl) ketone and 1H-heptane, with respect to the internal standard, perfluoro-cis-decalin, was studied using different negative ion chemical ionization (NICI) reagent gases. Under the NICI conditions used in our laboratory, methane was found to be the best NICI reagent gas for the optimization of the electron attachment (in terms of total ion current) for perfluorinated alkanes, alkenes, aromatics and 1-methyladamantane. Carbon dioxide and ammonia were found to be good NICI reagent gases for optimizing the response of hydrogen- and oxygen-containing fluorocarbons and highly symmetric PFCs such as perfluoroadamantane and perfluoro-trans-decalin. On the other hand, CF4 was found to yield the largest linear range in the calibration curve for perfluoro-C11-cycloalkanes (PCA) when perfluoro-cis-decalin was used as the internal standard. The employment of NH3 as the NICI reagent gas gave the most sensitive response to the increase of trace PCA concentration.
Metabolism of the antipsychotic drug tiospirone was studied in vitro with phenobarbital-induced rat liver microsomes. Metabolites were isolated and purified to homogeneity by high-performance liquid chromatography. It was possible to characterize the metabolites as trimethylsilyl (TMS) derivatives by gas chromatography/electron impact mass specrometry (GC/EIMS) so long as the sulfur was present in the reduced form. However, sulfoxide and sulfone analogs of tiospirone underwent reductive decomposition on the GC column. In addition, no molecular ions were observed in the EI spectra of these analogs. Desorption chemical ionization/mass spectrometry (DCI/MS) in the positive ion mode with methane as reagent gas successfully distinguished sulfoxides and sulfones from their parent sulfides. Protonated molecular ions were observed together with structurally significant fragment ions. Five microsomal metabolites of tiospirone were characterized by a combination of GC/EIMS and DCI/MS. From the structures of the metabolites three major pathways of metabolism were identified: N-dealkylation of the butyl side chain at the piperazinyl nitrogen, hydroxylation α to the glutarimidyl carbonyl at C-6 on the azaspirodecanedione ring, and sulfoxide formation on the benzisothiazole moiety.
Fast atom bombardment mass spectrometry was used in direct combination with enzymatic cleavages by endopeptidases for the identification of peptides derived from recombinant proteins. By this fast and simple reaction on the probe tip, the enzymes produced a mixture of smaller peptides. Molecular weights were calculated in advance and used for verification of the amino acid sequence of the original peptide as well as for partial structure determinations. Since the enzymatic reaction took place directly on the probe tip, sample material and time was saved. This technique may be applied especially in such cases where mass spectrometry alone does not give sufficient information and if the mass range of the instrument is limited, e.g. to 2000 daltons.
Disulphide bridges have been assigned in three different proteins by locating possible disulphide-linked peptides in enzymic digests of the proteins based on their molecular weight determined by plasma desorption mass spectrometry. Different strategies have been employed including in situ reduction of the nitrocellulose-bound peptides and confirmation of peptide identity by methyl esterification reactions or Edman degradation. The latter was needed for identification of glycosylated disulphide-linked peptides. For insulins cleavage between cysteine residues in close proximity was not possible; but a combination of molecular mass information, enzymic cleavage with two different enzymes and sequence analysis including identification of di-phenylthiohydantoin-cystine could ensure an unambiguous assignment of the disulphide bridges.
The least squares method for the solution of ion overlap problems in quantitative mass spectrometry first introduced by Brauman was used to quantify isotopic mixtures of chlorophenol derivatives. The samples analyzed consisted of the chlorophenols resulting from the cytochrome P-450 mediated oxidation of isotopic mixtures of chlorobenzenes, and were analyzed on a VG-7070 double-focusing mass spectrometer. The application of this method is described and its advantages are highlighted. In some instances the use of the least squares methods resulted in smaller standard deviations of the means of replicate analyses. Significant features of the technique include its simplicity of use, the universality of its application to all cases of ion overlap and its inherent ability to detect erroneous data, e.g. that due to the presence of impurities or mass spectral parameter variation.
The electrospray ionization (ESI) and plasma desorption (PD) mass spectra of over 20 peptides and proteins, with molecular weights (Mr) ranging between 1182 and 143000, have been directly compared. Both techniques produced molecular ions for the majority of materials studied; however, neither approach proved to be universally applicable. PD failed for a number of proteins that were successfully analyzed by ESI, including some of very high Mr. On the other hand, ESI failed for proteins that apparently could not acquire a sufficient number of positive charges to allow transmission through the quadrupole mass filter. A non-covalently bound adduct, ribonuclease S, did not survive either method intact and a simple glycoprotein, ribonuclease B, did not yield the expected molecular ion with either approach. The mass measurement accuracy of quadrupole ESI is five to tenfold better than obtained with a commercial time-of-flight PD mass spectrometer. Furthermore, ESI's superior mass resolution (with quadrupole mass spectrometers) will prove to be particularly helpful for the characterization of mixtures of closely related materials. Sensitivity was only compared qualitatively but is highly compound dependent with both techniques. In favorable cases, ESI spectra can be obtained on low femtomolar quantities of proteins while PD typically requires several hundred femtomoles to high picomoles, depending on a number of factors including Mr.
A new method for characterizing O-linked glycopeptides without chemical degradation is presented. Collisioninduced dissociation (CID) analysis of intact O-linked glycopeptides containing mono-and disaccharides was performed. For glycopeptides containing one hexose unit, both the peptide sequence and the site of attachment of the sugar moiety were obtained from a single high-energy CID spectrum. However, in a glycopeptide bearing multiple sugar residues per site, the CID spectrum was dominated by fragments resulting from cleavages of the carbohydrate substituents and the gas-phase deglycosylated peptide, thus obviating the concomitant observation of peptide sequence ions. Hence, information on the structures of the carbohydrate substituents was obtained, but not on the sites of attachment of these residues to the peptide. Subsequent CID analysis of the gas-phase deglycosylated peptide ion can be used to obtain the sequence of the peptide backbone from the same sample. This method holds promise for simultaneously determining the carbohydrate structure and the peptide sequence of intact O-linked glycopeptides without chemical degradation.
