Mass-spectrometric identification of trehalose 6-monomycolate synthesized by the cell-free system of Bacterionema matruchotii

The fluffy layer fraction prepared from Bacterionema matruchotii was found to possess high activity for the biosynthesis of mycolic acids which were bound to an unknown compound by an alkali-labile linkage [T. Shimakata, M. Iwaki, and T. Kusaka (1984) Arch. Biochem. Biophys. 229, 329-339]. To determine the structure of the mycolate-containing compound, it was purified and analyzed by field desorption (FD) and secondary ion mass spectrometry (SI-MS). When non-labelled palmitic acid was used as a precursor in the in vitro biosynthetic system, the underivatized product had a cationized molecular ion, [M + Na]+, at m/z 843 in FD-MS and a protonated ion, [M + H]+, at m/z 821 in SI-MS, corresponding to the quasimolecular ion of trehalose monomycolate (C32:0). In SI-MS, characteristic fragment ions due to cleavage of glycosidic linkages were clearly detected in addition to the molecular ion. If [1-13C]palmitic acid was the precursor, 2 mass unit increases in both the quasimolecular and fragment ions were observed, indicating that two molecules of palmitate were incorporated into the product. alpha-Trehalose was found in the aqueous phase after saponification of the product. By the electron impact mass spectrometry of the trimethylsilylated product, the mycolate was found to be esterified with an hydroxyl group at position 6 of the trehalose molecule. These results clearly demonstrated that the predominant product synthesized by the fluffy layer fraction with palmitate as substrate was 6-monomycolate (C32:0) of alpha-D-trehalose. Because newly synthesized mycolic acid was mainly in the form of trehalose monomycolate instead of free mycolate or trehalose dimycolate, the role of trehalose in the biosynthesis of mycolic acid is discussed.     

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.     

Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry.

Electrospray ionization (ESI) coupled with tandem mass spectrometry has been used in conjunction with microwave-mediated saponification, periodate oxidation, and clostridial sialidase hydrolysis to enable detailed structural characterization of gangliosides and their derivatives present in mullet milt. The gangliosides extracted from mullet milt were determined to be GM3, GM3 lactone, GM3 methyl ester, and 9-O-acetyl GM3. For the major ganglioside GM3 and all GM3 derivatives, the ceramide composition was revealed to be C18:1/C16:0. GM3 with a C18:0/C16:0 ceramide was also found as a minor ganglioside. Both the ganglioside intramolecular ester and the ganglioside methyl ester (lacking carboxylic acid groups) showed dominant chloride attachment peaks (M + Cl)- in negative ion ESI-MS in addition to low intensity peaks corresponding to (M-H)-. GM3 and O-acetyl GM3 bearing carboxylic acid functions showed only (M-H)-. In positive ion ESI, GM3 and O-acetyl GM3 revealed (M + 2Na-H)+ peaks in addition to (M + Na)+, indicating free exchange of the carboxylic acid proton with a sodium cation, while the ganglioside intramolecular ester and ganglioside methyl ester with no acidic protons yielded only (M + Na)+. The strategy of employing ESI-MS to detect products of established wet chemical reactions represents a general approach for elucidation of ganglioside structural details.     

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.     

The glycosphingolipid composition of the human hepatoma cell line,Hep-G2

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM3, and lactosyl sulfatide. Four additional minor components (3-fucosyl-lactosamine containing glycolipids, asialo GM2, galactosylgloboside, and ganglioside GM1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.     

Secondary ion mass spectrometry for sulfoglycolipids: application of negative ion detection

A series of underivatized sulfoglycolipids (SM4g, lyso-SM4g, SM4s, SM3, SM2, SB2, and SB1a) from various tissues were analyzed by both positive (POS-SI-MS) and negative (NEG-SI-MS) secondary ion mass spectrometry. By POS-SI-MS were detected the molecular ions of sulfoglycolipids in the form with sodium or potassium together with some fragment ions useful for the carbohydrate sequence determination. The analysis of monosulfogangliotriaosyl- or monosulfogangliotetraosylceramide and bis-sulfoglycolipid was difficult due to noise in the high mass region. On the other hand, NEG-SI-MS of sulfoglycolipids gave more intense signals from molecular ion of (M-H)- for monosulfoglycolipids and [M-H+Na)-H)- for bis-sulfoglycolipid. Many fragment ions useful for the elucidation of the carbohydrate sequences were also obtained with significant intensities. The fragmentation was assessed to occur at the glycosidic linkages to form ions of the oligosaccharides with or without ceramide. These ions were useful for sugar sequencing and also for distinguishing the differences in the position of the sulfate group. The intensities of saccharide ions without sulfate were lower than those with sulfates. In the case of SB2 and SB1a, containing 2 mol of sulfate ester groups, the molecular ion was detected as [M-H+Na)-H)-. Also, fragment ions with 2 mol of sulfate were detected as the sodium-additive form. It was concluded that NEG-SI-MS is a very useful technique for the structural elucidation of higher sulfoglycolipids.     

Glycosphingolipid specificity of the human sulfatide activator protein

The interaction of the sulfatide activator protein with different glycosphingolipids have been studied in detail. The following findings were made. 1. The sulfatide activator protein forms water-soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe-Seyler's Z. Physiol. Chem. 356, 6588-6591] and various other glycospingolipids. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules. 3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein. 4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by beta-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.     

Structural Analyses of Xyloglucan Heptasaccharide by the Post-source Decay Fragment Method Using Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

In the post-source decay (PSD) fragment spectrum of a reduced xyloglucan heptasaccharide (XXXGol) from tamarind seeds, eleven sodium-adduct fragment ions and a precursor ion [M + Na]⁺ were clearly observed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Each fragment ion interval corresponded to the absence of unhydroxylose, unhydroglucose, and glucitol residues, indicating that PSD fragmentation cleavage in the sugar compound occurred only at glycosidic linkages close to the oxygen atom of saccharide ring members, and not in inner sugar ring bonds. The PSD fragment ions were classified into two series, one involving the reducing end and the other involving the non-reducing end. Structural information from both the reducing and non-reducing ends could therefore be simultaneously obtained from the measurement of the positive ion mode. Almost all the fragment ions from species larger than trisaccharide residues could be detected in this PSD fragment experiment. Such fragmentation information will enable the structural determination of xyloglucan oligosaccharides.     

Effect of calcium ions on the thermotropic behaviour of neutral and anionic glycosphingolipids

In the concentration range of 10(-5) to 10(-1) M Ca2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-GM1, sulfatide, GM1, GD1a, GT1b) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca2+. The more marked effect of Ca2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca2+ between 10(-5) and 10(-3) M (up to a molar ratio of Ca2+/sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10(-1) M Ca2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.     

Proton nuclear magnetic resonance of neutral and acidic glycosphingolipids

A number of intact neutral glycosphingolipids (globo, asialoganglio, neolacto, and gala series), gangliosides, and sulfatide were analyzed by proton nuclear magnetic resonance (NMR) using dimethyl-d6 sulfoxide as a solvent at different conditions of measurement. The chemical shifts of amide proton of ceramide, N-acetylhexosamine and sialic acid moieties were positioned with regularity, thus providing their molar composition. The chemical shifts of amide proton in ceramide moiety differed with respect to constituent fatty acids; delta 7.45 to 7.52 ppm at 25 degrees C for the nonhydroxy acids and 7.32 to 7.42 ppm for the hydroxy acids. The chemical shifts of methyl proton in N-acetyl group were distinguished between N-acetylhexosamine and N-acetylneuraminic acid, and those in N-acetylgalactosamine were discriminated between neutral glycolipids and gangliosides. In the presence or absence of D2O in dimethyl sulfoxide at 110 degrees C, the anomeric protons resonated with regularity characteristic of respective monosaccharide linkages, and the anomeric protons of N-acetylgalactosamine in neutral glycolipids and gangliosides were clearly distinguished. The present study thus demonstrates the general applicability of NMR procedure to glycosphingolipids, providing the determination of chemical composition of both the lipophilic and carbohydrate moieties and the structural elucidation.     

Thermospray HPLC/MS: a new mass spectrometric technique for the profiling of steroids

The analysis of various steroid classes by thermospray HPLC-MS using solvent systems containing 0.1 M ammonium acetate has been described. For simple unconjugated 3-oxo-4-ene steroids the positive ion spectra are dominated by a parent ion M + H+ and with increasing numbers of hydroxyl group intense ions formed by sequential losses of water (M + H- n18)+ become important. Steroids with dihydroxyacetone side-chains readily lose these side-chains and the resulting (M + H-60)+ fragment is the base peak in their spectra. The (M + H-60)+ ion is not important for most steroids with glycerol-type side-chains. Although competition between thermal degradation and vaporization was observed at lower concentrations, the effect was minimized after optimizing conditions and the protonated molecular ion was easily detected when as little as 1-10 pmol of material were injected on-column. Steroid glucuronides when analyzed in the negative ion mode give simple spectra with base peak and parent ion (M-H)-. Lack of fragmentation permits facile and sensitive measurement of individual glucoronides by selected-ion-monitoring. Extensive fragmentation is seen in the positive ion mode with sequential losses of H2O from the molecular ions (M + NH4)+ and from the aglycone fragment ion. For simple unconjugated steroids the sensitivity of HPLC-MS in selected-ion-monitoring mode can be excellent. When the protonated molecular ion of testosterone was monitored the signal/noise ratio for 30 pg testosterone was about 10.     

Fish egg polysialoglycoproteins: structures of new sialooligosaccharide chains isolated from the eggs of Oncorhynchus keta (Walbaum). Fucose-containing units with oligosialyl groups

A series of acidic oligosaccharide alditols having different neutral core oligosaccharides were isolated from salmon egg polysialoglycoproteins by alkali-borohydride treatment followed by anion-exchange chromatography and Iatrobead chromatography. Their structures were determined by methylation analysis, molecular secondary ion mass spectrometry of underivatized oligosaccharides, and enzymatic desialylation. The molecular secondary ion mass spectra of intact sialooligosaccharides exhibit pronounced quasi-molecular-ion peaks, (M + H)+, (M + Na)+, (M + 2Na - H)+, and/or (M + K)+, as well as some diagnostic sequence ion peaks. Of a number of oligosaccharide alditols, the following are novel: Fuc alpha 1 leads to 3GalNAc beta l1 leads to 3Gal beta 1 leads to 4Gal beta 1 leads to 3[(leads to 8NeuGc alpha 2)n leads to 6]GalNAcol (n = 1-6). The proton nuclear magnetic resonance spectra of these oligosaccharides are also reported and discussed.     

Fast atom bombardment and tandem mass spectrometry for structure determination of steroid and flavonoid glycosides

The combination of fast atom bombardment (FAB) and tandem mass spectrometry (MS-MS) was tested for its applicability to generate useful structural information for steroid and flavonoid glycosides. The following compounds were investigated: quercetin, myricitrin, apigetrin, fraxin, rutin, neohesperidin, hesperidin, naringin, apiin, cymarin, digoxin, digitoxin, xanthorhamnin, and frangulin. Upon FAB, the sample molecules are desorbed as (M + H)+, (M - H)-, or as (M + Na)+ or (M + K)+. Collisional activation of (M + H)+ or (M - H)- ions in the MS-MS experiment leads to sequential losses of glycoside moieties in a manner which permits the sequence of glycosides to be established. Some glycosides occur as mixtures of homologs. Proper interpretation of the MS-MS or collisional activation decomposition spectra often allows the homology to be located. In addition to the simple and highly selective fragmentations observed in this combined experiment, FAB and MS-MS also remove interference caused by the ubiquitous matrix ions which are desorbed by FAB.     

Analysis of oxidative warfarin metabolites by thermospray high-performance liquid chromatography/mass spectrometry

Oxidative metabolites of the anticoagulant, warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one], produced by the actions of cytochromes P450 were analyzed by thermospray high-performance liquid chromatography/mass spectrometry. Warfarin, dehydrowarfarin, and the 6-, 7-, 8-, and 4'-hydroxy derivatives of warfarin were found to ionize well by the thermospray process in the presence of ammonium acetate. Thermospray mass spectra of these compounds were generally dominated by the protonated molecule, (M + H)+, and ions formed by the loss of water from the protonated molecule, (M + H - H2O)+. Fragment ions arising from the hydroxycoumarin, benzylhydroxycoumarin, and phenylbutanone portions of the molecules were observed, and the relative intensity of these fragment ions was greatly increased with filament ionization and application of a high repeller potential (100-130 V). Selected-ion monitoring of the (M + H)+ and (M + H - H2O)+ ions provided sensitivities for these compounds in the 2 to 10 ng range. A method employing thermospray HPLC/MS with selected-ion monitoring and internal standard quantitation for the analysis of the oxidative metabolites of warfarin is described.     

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.     

Atmospheric pressure photoionization mass spectrometry of per-O-methylated oligosaccharides related to D-xylans

Three d-xylan type per-O-methylated trisaccharides with various types of linkages between the d-xylopyranose units were examined by atmospheric pressure photoionization (APPI) mass spectrometry in the positive ion mode. The most interesting feature of a thermospray mass spectrum using the APPI source with UV lamp switched off, is the exclusive production of [M+Na]⁺ adduct ions. [M+Na]⁺ cationized ions are the most abundant species in the case of APPI mass spectrometry. The second ionization process has no analogy in the case of substances studied using APPI to date. This aspect involves the addition of a water molecule to the molecular ion of a per-O-methylated saccharide, giving rise to [M+H₂O]⁺ adduct ions. The [M+H₂O]⁺ species are readily detected at m/z 544, and are clearly visible for all three isomers studied. The MS/MS spectrum of [M+Na]⁺ ions contains a base peak at m/z 375, produced by a Y-type cleavage of the trisaccharide, along with a hydrogen rearrangement on the terminal interglycosidically linkage glycosidic oxygen atom. The [M+H₂O]⁺ species fragment largely give rise to ions at m/z 175, 143 and, as a result, the m/z 111 ion is unique to nonreducing terminal units.     

Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis.     

Characterization of Neutral and Acidic Glycosphingolipids in Brains of Two Patients with GM1 Gangliosidosis Type 1 and Type 2

Brains of two patients with GM1 gangliosidosis type 1 and type 2, together with the age-matched control brains, were analyzed for glycosphingolipids. Six species of neutral glycolipids, eight species of gangliosides, and sulfatide were isolated from the diseased brains and identified. In addition to GM1 ganglioside and its asialo derivative, the diseased brains accumulated considerable amounts of gangliotriaosylceramide and glycolipids belonging to the globo series, the accumulation of which cannot be explained by deficient beta-galactosidase activity in this disease. GM4 ganglioside was detected in the type 2 brain, but not in type 1. As to fatty acid composition of monohexosylceramides and sulfatide in the two diseased brains, stearic acid was more predominant in the type 1 brain than in the type 2 brain. In light of our previous observations on a Tay-Sachs brain and present results, it appears that metabolism of the globo series glycolipids, which is active in normal brain at early infancy but inactive thereafter, remains in brains with GM1 gangliosidosis (types 1 and 2) and Tay-Sachs disease, reflecting a disturbance in development of the brain.     

Human annexin V binds to sulfatide: contribution to regulation of blood coagulation

Annexin V is a calcium-dependent phospholipid-binding protein that exhibits anticoagulant activity on binding to phosphatidylserine exposed on the activated surfaces of endothelial cells and platelets, inhibiting activation of factor X and prothrombin in the blood coagulation cascade. Sulfatide (galactosylceramide I(3)-sulfate), one of the glycosphingolipids of the platelet cell membrane, is thought to be involved in blood coagulation systems via activation of factor XII. In this study, we examined whether or not annexin V binds to sulfatide and affects the coagulant activity of sulfatide. Solid phase assaying of annexin V revealed that it binds specifically to sulfatide, i.e. not to galactosylceramide or gangliosides, in the presence of calcium ions. Affinity analysis by means of surface plasmon resonance showed that the K(D) of the interaction between annexin V and sulfatide is 1.2 micro M. Kinetic turbidometric assaying of plasma coagulation initiated by CaCl(2) revealed that the coagulation rate in the presence of sulfatide or phosphatidylserine was decreased by annexin V. These results suggest that annexin V regulates coagulability in the blood stream by binding not only to phosphatidylserine but also to sulfatide.     

A computational study of structure-reactivity relationships in Na-adduct oligosaccharides in collision-induced dissociation reactions

Elucidating the fragmentation mechanisms in oligosaccharides using theoretical calculations is useful in analyzing the experimentally obtained mass spectra. Semi-empirical and ab initio quantum mechanics calculations were used to study the relationship between the structure and reactivity and the chemical properties of oligosaccharides. In these calculations, sodium-cationized oligosaccharides were investigated to determine Na+ ion affinity at several binding positions; in addition, the dependence of the glycosidic bond cleavage on the Na+ position was examined. The calculated structures reported in this study are directed at interpreting experimentally observed fragment ions, resulting from the cleavage of the glycosidic bonds. The calculated results for oligosaccharides containing between three and five monosaccharide units (27 oligosaccharides) were compared with experimental data generated by matrix-assisted laser-desorption/ionization (MALDI) using a quadrupole ion trap (QIT) with a time-of-flight (TOF) mass spectrometer (MS).