Betaines of alfalfa : characterization by fast atom bombardment and desorption chemical ionization mass spectrometry

Leaf tissue of alfalfa (Medicago sativa L.) was found to contain prolinebetaine, pipecolatebetaine, hydroxyprolinebetaine, and glycinebetaine. As n-butyl esters, these chemical species exhibit molecular cations at mass/charge ratio (m/z) 200, 214, 216, and 174, respectively, when analyzed by fast atom bombardment mass spectrometry. The underivatized betaines exhibit protonated molecular ions at m/z 144, 158, 160, and 118, respectively, when analyzed by desorption chemical ionization mass spectrometry. Extensive (>45-fold) genotypic variation for hydroxyprolinebetaine level was identified in alfalfa. Because a significant inverse correlation between prolinebetaine and hydroxyprolinebetaine levels was observed among 15 alfalfa genotypes evaluated, it is possible that these compounds may be derived from a common intermediate. Birdsfoot trefoil (Lotus corniculatus L.) contained prolinebetaine, but only traces of glycinebetaine, pipecolatebetaine, and hydroxyprolinebetaine. Red clover (Trifolium pratense L.) lacked prolinebetaine, pipecolatebetaine, and hydroxyprolinebetaine, but contained appreciable levels of both glycinebetaine and trigonelline. Trigonelline was not detectable in the leaf tissue of any alfalfa genotype or cultivar evaluated.     

Analysis of long-chain bases in sphingolipids by positive ion fast atom bombardment or matrix-assisted secondary ion mass spectrometry

The structures of long-chain bases are expressed as [CH2C(NH2) = CHR]+ (Z+) in the positive ion mode spectra obtained on fast atom bombardment (FAB) mass spectrometry or liquid-matrix-assisted secondary ion mass spectrometry (SIMS) [Benninghoven, A., Ed. (1983) Ion Formation from Organic Solids, Springer, Berlin]. This phenomenon is common to sphingolipids in general: glycosphingolipids [see reviews by Sweeley and Nunez [Sweeley, C. C., & Nunez, H. A. (1985) Annu. Rev. Biochem. 54, 765] and Kanfer and Hakomori [Kanfer, J. N., & Hakomori, S. (1983) Handb. Lipid Res. 3]] and phosphonosphingolipids [Hayashi, A., & Matsubara, T. (1982) in New Vistas in Glycolipid Research (Makita, A., Handa, S., Taketomi, T., & Nagai, Y., Eds.) p 103, Plenum, New York], inclusive. Phytosphingosine compounds show the same type of fragmentation without additional dehydration if a neutral matrix is used. A Z+ ion is easily detected in the lower mass region (m/z 200-400) as an even mass number fragment ion, and confirmation is made by means of B/E constant and B2/E constant linked scan techniques [Boyd, R. K., & Beynon, J. H. (1977) Org. Mass Spectrom. 12, 163; Boyd, R. K., & Shushan, B. (1981) Int. J. Mass Spectrom. Ion Phys. 37, 355; Macdonald, C. G., & Lacey, M. J. (1984) Org. Mass Spectrom. 19, 55]. [Principles of linked scannings are explicitly summarized by Jennings and Mason [Jennings, K. R., & Mason, R. S. (1983) in Tandem Mass Spectrometry (McLafferty, F. W., Ed.) p 197, Wiley, New York] besides the cited literature.]     

Positive ion fast atom bombardment mass spectrometry of some small oligosaccharides

Positive ion fast atom bombardment mass spectrometry appears to be a very useful method for the determination of molecular weight and composition of underivatized di- and trisaccharides. Information on the identity of the monosaccharide units can be obtained from the metastable ion and collisional activation spectra of selected ions. The type of linkage between the monosaccharides is reflected in some spectral characteristics, but the differences are relatively small and do not always allow an unambiguous identification. The position of a fructose unit in a trisaccharide molecule is shown by the collisional activation spectra of the [M + H]+ ion, as an anhydrofructose molecule is easily eliminated from the ions in which fructose is in a terminal position.     

Observations on the presence of oligomeric species in the positive ion fast atom bombardment mass spectra of mixed ligand complexes of magnesium

Attention is drawn to the occurrence of oligomeric ions in the FAB-MS of a group of mixed ligand complexes of magnesium; possible mechanisms of formation are suggested.     

Coaxial continuous flow fast atom bombardment for higher-molecular-weight peptides: comparison with static fast atom bombardment and electrospray ionization.

A comparison of coaxial continuous flow fast atom bombardment (FAB) with static FAB and with electrospray ionization (ESI) for the analysis of 'high'-mass peptides (Mr = 3000-4000) is presented. Sensitivities of the peptides by coaxial continuous flow FAB is nearly an order of magnitude better than by static FAB. Single-scan spectra with good signal-to-noise can be obtained from as little as 200 fmol (by flow injection analysis). Detection limits by ESI mass spectrometry were found to be equivalent to 20 times higher than by coaxial continuous flow FAB on a per mole basis, but 4-20 times lower on a concentration basis, owing to the greater flow per unit time employed in the ESI mass spectrometric experiments.     

1-trans-Parinaroyl phospholipids: Synthesis and fast atom bombardment/electron impact mass spectrometric characterization

1-trans-Parinaroyl-2-linoleoyl-sn-glycero-3-phosphocholine (1–18:4-2-18:2-GPC) was synthesized from lecithin and parinaric acid by the following route: $\text{diacyl-GPC}\text{→}\text{GPC}\text{→ 1,2-}\text{di}\text{-18:4-}\text{GPC}\text{(}\text{I}\text{) → 1–18:4-}\text{GPC}\text{(}\text{II}\text{) → 1–18:4-2-18:2-}\text{GPC}\text{(}\text{III}\text{)}$. The identity of I, II and III was established by fast atom bombardment (FAB) mass spectrometry of the intact molecules as well as electron impact (E1) mass spectrometry of the corresponding O-TMS derivatives obtained after phospholipase C treatment and silylation. Temperature dependent phase transition of phospholipid liposomes was performed in the presence of III.     

Analysis of muramic acid in holocene microbial environments by gas chromatography,electron impact,and fast atom bombardment mass spectrometry

Analytical procedures have been modified to determine the abundance of muramic acid in four different Holocene sediment samples. Muramic acid is specific to the peptidoglycan moiety of the cell walls of most eubacterial pro‐karyotic organisms. The following procedure seemed to be the most appropriate for the detection of muramic acid and amino acids, including diaminopimelic acid. Hydrolysis of the samples (in 6 N HCl, 4.5 h, at 100°C) was followed by separation and purification of amino sugars and amino acids using Amberlite XAD‐2 and then Bio‐Rad AG 50W‐X8 resins. The N,O‐heptafluorobutyryl‐n‐butyl ester derivatives were prepared by esterification in acidified (3 N HCl) n‐butanol for 3 h at 100°C, followed by acylation by refluxing with heptafluorobutyric anhydride in acetonitrile (2:1 v/v) for 12 min at 150°C. The derivatives were analyzed by gas chromatography (GC) and gas chromatography‐mass spectrometry. Fast atom bombardment (FAB) ionization was used for the muramic acid derivative to determine its molecular weight and structure, d‐and l‐amino acids were separated by GC and a capillary chiral column. By using this technique a stable N,O‐heptafluo‐robutyryl‐n‐butyl ester derivative of muramic acid was identified at picogram levels in Holocene sedimentary microbial communities. It has been reported previously that microorganisms in sediments rapidly degrade muramic acid from cell walls of dead prokaryotes. Kinetic experiments revealed that muramic acid was relatively stable in intact cell walls but decomposed rapidly in the free form. These investigations noted above showed that the concentration of muramic acid may be used as an indicator of the presence of the intact cell walls of cyanobacteria and most other bacteria in Holocene microbial communities, and of microbial contamination in samples older than the Holocene.     

Negative ion ammonia chemical ionization and electron impact ionization mass spectrometric analysis of steryl fatty acyl esters

Synthesis of steryl palmitates, varied in the nature of the steryl moiety, provided model compounds for investigation of the mass spectrometric behavior of steryl long-chain fatty acyl esters. The structure of the steryl moiety was varied according to: (i) position and degree of unsaturation in the steroid nucleus and C-17 side-chain, (ii) position and degree of methylation, (iii) presence or absence of a 9 beta, 19-cyclopropane ring. Compounds were chosen so as to be representative of biochemically important steryl esters. Electron impact (EI) behavior of steryl palmitate esters closely resembles that of their short-chain (e.g. acetate) counterparts. M+.ions were generally weak or absent and the major high mass ions arose from characteristic fragmentations of the steroid nucleus following loss of the acyl moiety ([M-RCO2H]+.). Fragment ions characteristic of the acyl moiety were lacking. Negative ion chemical ionization (NICI) using ammonia as reagent gas, on the other hand, afforded spectra containing characteristic fragment ions [RCO2]-, [RCO2-18]-, and [RCO2-19]- from which the nature of the fatty acyl moiety can be readily deduced. Hence, NICI and EI provide complementary means of ionization for the mass spectrometric determination of structures of steryl esters.     

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.     

Reaction mechanism and fragment ion structure determination of deprotonated small oligosaccharides, studied by negative ion fast atom bombardment (tandem) mass spectrometry

The negative ion mass spectrometric characteristics of a series of di- and trisaccharides and the tetrasaccharide stachyose have been studied using fast atom bombardment mass spectrometry. The molecular weight of the compounds can easily be derived from their mass spectra, which all show an abundant [M - H]- ion peak. The application of metastable ion and collisional activation techniques to selected pseudomolecular and fragment ions appears to be appropriate for the determination of the position and anomeric type of linkage in the molecules, and provides information concerning the monosaccharide units involved. Important fragmentation reactions have been traced and reaction mechanisms, supported by deuterium labelling experiments, are proposed. An experiment describing the application of the findings of this study to a glycosphingolipid molecule demonstrates its potential value for biological systems.     

Some studies on the negative ion fast atom bombardment mass spectroscopy of nucleic acids

The effect of organic bases and crown ethers on the enhancement of the intensities of negative molecular ions of nucleic acids are discussed.     

Mass-analysed ion kinetic energy spectra and B1E-B2 triple sector mass spectrometric analysis of phosphoinositides by fast atom bombardment

Fast atom bombardment is shown to produce useful spectra of the three phosphoinositides and the metabolically related phospholipids, lysophosphatidylinositol and phosphatidic acid. Analysis of the [M-H]- ions for fatty ester composition by mass-analysed ion kinetic energy spectra (MIKES) is shown to be inadequate to resolve fatty acyl daughter ions when the parent ion contains isobaric species. However, analysis on a triple sector instrument with and without collisional activation does provide complete compositional information. Quantitative analysis of the fatty ester content of each lipid molecular species is complicated by dissimilar ion yields from fatty acyl-bearing fragments from compositionally different parent ions.     

Identification and quantification of mutagenic halogenated cytosines by gas chromatography,fast atom bombardment,and electrospray ionization tandem mass spectrometry

Oxidative modification of nucleic acids has been implicated in carcinogenesis. One potential mechanism involves halogenation by the myeloperoxidase and eosinophil peroxidase systems of phagocytes. In the current studies, three mass spectrometric methods for the in vitro and in vivo analysis of halogenated cytosines and deoxycytidines were compared: gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) with a quadrupole instrument, fast atom bombardment or electrospray ionization (ESI) tandem MS with a four-sector magnetic instrument, and liquid chromatography ESI tandem MS (HPLC-ESI-MS/MS) with an ion-trap instrument. GC-EI-MS with selected ion monitoring of dimethyl-tert-butylsilyl derivatives of nucleobases was the most sensitive method. High-energy collisionally induced dissociation MS/MS analysis with a four-sector magnetic instrument yielded detailed structural information about halogenated nucleoside adducts but required relatively large amounts of material. The most sensitive analysis of intact halogenated deoxycytidine was achieved with extracted ion chromatograms using HPLC-ESI-MS/MS with an ion-trap instrument. Our results indicate that GC-EI-MS is the methodology of choice for ultrasensitive analysis of halogenated cytosines. HPLC-ESI-MS/MS provides greater structural detail for these compounds and may rival GC-EI-MS in sensitivity with more advanced liquid chromatography applications. The mass spectrometric methods we have developed should be useful for evaluating the role of phagocyte-derived oxidants in halogenating nucleobases, nucleosides, and DNA at sites of inflammation.     

Chemical ionization and electron impact mass spectra of oligosaccharides derived from sphingoglycolipids

Chemical ionization (CI) mass spectra with isobutane and ammonia for the oligosaccharides obtained from sphingoglycolipids were compared with their electron impact (EI) mass spectra. The oligosaccahride moieties were liberated from the parent glycolipids and were further reduced with sodium borohydride. They were analyzed as their permethyl peracetyl and pertrimethylsilyl derivatives. In the CI spectra, peaks corresponding to QM+ and/or [M-59]+ were observed in all of the peracetylated oligosaccharides examined. In CI with ammonia as the reagent, H+ was transferred to nitrogen-containing saccharides to produce [MH]+ and NH4 was transferred to nitrogen-free saccharides to yield [M+NH4]+ as QM+. Non-reducing ends yielded very intense peaks in CI spectra. On the other hand, the reduced end, glucitol, produced rather prominent peaks in EI spectra. Fragment ions due to cleavage of glycosidic bonds were major ones under the CI conditions, and they could be used for elucidating the sugar sequence in the oligosaccharides. An additional characteristic feature in the CI spectra was that ions due to scission of hexosaminyl glycosidic linkages were observed with very high intensities.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

The analysis of multiple O-phosphoseryl-containing peptides by fast atom bombardment mass spectrometry

Summary Positive and negative ion FAB mass spectrometry were found to be useful for the structural analysis of phosphorylated peptides containing multiple O-phosphoseryl residues. The positive ion FAB mass spectra obtained for Ac-Ser(P)-Ser(P)-NHMe and Ac-Ser(P)-Ser(P)-Ser(P)-NHMe showed that -eliminative loss of H₃PO₄ from the Ser(P)-residue was a major event in the fragmentation of the two phosphopeptides and that successive losses of H₃PO₄ from the [M+H]⁺ ion occurred when the Ser(P)-cluster was located at the N-terminus. In contrast, the FAB mass spectrum of Ac-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe showed only a single loss of H₃PO₄ from the [M+H]⁺ ion, with further losses of H₃PO₄ from internal Ser(P)-residues only occurring when fragmentation of the parent phosphopeptide generated daughter fragments that contained (part of) an N-terminal Ser(P)-residue. Negative ion FAB mass spectrometry also proved useful for the structural analysis of the three Ser(P)-peptides and showed high-intensity [M-H]^- ions along with minor [M-H-80]^- fragment ions.Abbreviations Ac acetyl - Ala dehydroalanyl - FAB-MS fast atom bombardment mass spectrometry - LSIMS liquid secondary ion mass spectrometry - NHMe N-methylamide - Ser(P) O-phosphoseryl - Thr(P) O-phosphothreonyl     

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, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol-3-0-beta-D-glucuronide, present in cerebrotendinous xanthomatosis (CTX) patients, was investigated by positive ion fast atom bombardment mass spectrometry (FAB-MS). The spectrum was characterized 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 in the rapid diagnosis of CTX.     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.