Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry

Glycosphingolipids and gangliosides have been investigated by using fast atom bombardment high-performance tandem mass spectrometry (FABMS/MS). Homologous compounds have been investigated in order to ascertain the fragmentation. Collision-induced dissociation spectra of the molecular species in the positive ion mode mainly afford information on the ceramide constitution (aglycon as a whole, N-acyl residue, and long-chain base), whereas negative ion spectra show fragments informative of the sugar sequence and the degree of branching of the carbohydrate. In the case of gangliosides carrying a complex oligosaccharide moiety, collision spectra of fragment ions have been performed in order to gain additional structural data. The advantage of tandem mass spectrometry over conventional fast atom bombardment mass spectrometry (FABMS) consists in the fact that collision spectra of the individual components from mixtures, as usually encountered with these kinds of samples, can be recorded. Furthermore, the exclusion of most of the interfering signals from the matrix allows the identification of pertinent fragments at low mass.     

Gangliosides as markers of cortisone-sensitive and cortisone-resistant rabbit thymocytes: characterization of thymus-specific gangliosides and preferential changes of particular gangliosides in the thymus of cortisone-treated rabbits

Neutral glycosphingolipids and gangliosides in rabbit thymus, spleen, bone marrow, and erythrocyte ghosts were analyzed by conventional chemical and enzymatic procedures and negative ion fast atom bombardment mass spectrometry (FABMS). Thymus gangliosides showed a characteristic composition. Major gangliosides comprising 75% of the total thymus gangliosides were sialosyl lacto-N-neo-tetraosyl- and sialosyl lacto-N-nor-hexaosylceramides containing NeuGc and palmitic acid. These major thymus gangliosides were not detected in spleen, bone marrow, or erythrocytes, whereas GD1a, which was not present in the thymus even in a trace amount, was present in spleen and bone marrow. In addition, the major gangliosides in rabbit thymus were preferentially reduced when an animal was given an intraperitoneal injection of cortisone acetate, as found on analysis 48 h later. The decrease was accompanied by a concomitant increase in NeuAc-containing GM3 with longer chain fatty acids.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

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.     

The first replacement of a chlorosulphonyloxy group by chlorine at C-2 in methyl α-D-glucopyranoside and sucrose derivatives

Treatment of methyl 3-O-acetyl-4,6-O-benzylidene-α-D-glucopyranoside 2-chlorosulphate (2), 3,4,6,3′,4′,6′-hexa-O-acetylsucrose 2,1′-bis(chlorosulphate), 3,4,6,3′,4′,6′-hexa-O-acetyl-1′-O-benzoylsucrose 2-chlorosulphate, and 3,4,3′,4′-tetra-O-acetyl-6,6′-dichloro-6,6′-dideoxysucrose 2,1′-bis(chlorosulphate) with lithium chloride in hexamethylphosphoric triamide gave the corresponding chlorodeoxy-manno derivatives. Treatment of the 2-chlorosulphate 2 with such nucleophilic reagents as lithium bromide, sodium azide, sodium chloride, and sodium benzoate in hexamethylphosphoric triamide gave the 2-hydroxy compound as a major product. Selective chlorination at C-1′ was achieved when 3,4,6,3′,4′,6′-hexa-O-acetylsucrose was treated with sulphuryl chloride in a mixture of pyridine and chloroform.     

beta-D-Galactoside transport in Escherichia coli. Reversible inhibition by Aprotic Solvents and its Reconstitution in transport-negative membrane vesicles.

At relatively low concentrations (less than 3M) the aprotic solvents: dimethylsulfoxide, N-methylpyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited beta-D-galactoside transport by whole cells, and the derived membrane vesicles of Escherichia coli. Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure...     

Improved synthesis of cord factor analogues via the Mitsunobu reaction

Treatment of trehalose with triphenylphosphine, diisopropyl azodicarboxylate and beta-O-tetrahydropyran-2-ylmycolic acid in 1:1 hexamethylphosphoric triamide/dichloromethane, followed by removal of the tetrahydropyranyl protecting group, gave cord factor (1) in good yield, under exceptionally mild conditions. Two new cord factor analogues were similarly prepared from beta-O-methylmycolic acid and from the alpha, beta-unsaturated 'anhydro' mycolic acid respectively. The procedure, employing excess trehalose, can also be used for the synthesis of trehalose monomycolates in good yield. No protection of the carbohydrate is required.     

Localization and imaging of gangliosides in mouse brain tissue sections by laserspray ionization inlet

A new ionization method for the analysis of fragile gangliosides without undesired fragmentation or salt adduction is presented. In laserspray ionization inlet (LSII), the matrix/analyte sample is ablated at atmospheric pressure, and ionization takes place in the ion transfer capillary of the mass spectrometer inlet by a process that is independent of a laser wavelength or voltage. The softness of LSII allows the identification of gangliosides up to GQ1 with negligible sialic acid loss. This is of importance to the field of MS imaging, as undesired fragmentation has made it difficult to accurately map the spatial distribution of fragile ganglioside lipids in tissue. Proof-of-principle structural characterization of endogenous gangliosides using MS(n) fragmentation of multiply charged negative ions on a LTQ Velos and subsequent imaging of the GD1 ganglioside is demonstrated. This is the first report of multiply charged negative ions using inlet ionization. We find that GD1 is detected at higher levels in the mouse cortex and hippocampus compared with the thalamus. In LSII with the laser aligned in transmission geometry relative to the inlet, images were obtained in approximately 60 min using an inexpensive nitrogen laser.     

Solution-phase secondary-ion mass spectrometry of protonated amino acids

Although sulfolane proved unexpectedly to be a poor solvent for solution-phase secondary-ion mass spectrometry of underivatized amino acids in the presence of thallium(I) salts, glycerol was somewhat more effective. Also, the addition of trifluoromethanesulfonic acid proved more effective than addition of the metal in generating molecular ion complexes. A convenient and reliable method for rapidly determining amino acid molecular ions is based on these observations.     

Syntheses stereoselectives de 1-thioglycosides

2,3,4,6-Tetra-O-acetyl-β-d-mannopyranosyl chloride (2) was obtained in 70% yield by the action of lithium chloride on 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide (1) in hexamethylphosphoric triamide. p-Nitrobenzenethiol reacted with 1 and 2 as well as with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (9) or its β-d-chloro analog (10), giving exclusively and in good yield the corresponding p-nitrophenyl 1-thioglycosides of inverted anomeric configuration. The 1,2-cis-d-manno and -glucop-nitrophenylglycosides were likewise prepared. α-d-Glucopyranosyl 1-thio-α-d-glucopyranoside was similarly obtained by the action of the sodium salt of 1-thio-α-d-glucopyranose on the β-chloride 10 in hexamethylphosphoric triamide, or by treatment of 10 with sodium sulfide, with subsequent deacetylation. Analogous procedures allowed the preparation of β-d-mannopyranosyl 1-thio-β-d-mann opyranoside, the corresponding α,β anomer and α-d-glucopyranosyl 1-thio-α-d-mannopyranoside, starting from bromide 1, 1-thio-α-d-mannopyranose (8),and chloride 10, respectively. When acetone was used as solvent, the reaction between 1 and 8 led instead to the α,α anomer. The thio disaccharides that are interglycosidic 4-thio analogs of methyl 4-O-(β-d-galactopyranosyl)-α-d-galactopyranoside, methyl α-cellobioside, and methyl α-maltoside, respectively, were obtained by way of the peracetates of methyl 4-thio-α-d-galactopyranoside and -glucopyranoside by reaction of the corresponding thiolates with tetra-O-acetyl-α-d-galactopyranosyl bromide, bromide 9, or chloride 10, respectively, in hexamethylphosphoric triamide. These 1-thioglycosides, and (1→1)- and (1→4)-thiodisaccharides, were characterized by ¹H- and ^{1 3}C-n.m.r. spectroscopy. Correlations were established between the polarity of the sulfur atom and certain proton and carbon chemical-shifts in the 1-thioglycosides in comparison with the O-glycosyl analogs; these correlations permitted in particular the unambigous attribution of anomeric configuration.     

The synthesis and the 1h- and 13c-nuclear magnetic resonance spectroscopy of the cyclic sulfites of some sugars

Treatment of methyl β-D-ribofuranoside with thionyl chloride in hexamethyl-phosphoric triamide gives two diastereoisomeric methyl 5-chloro-5-deoxy-β-D-ribo-furanoside 2,3-cyclic sulfites. Similar cyclic sulfites are formed from benzyl β-D-ribofuranoside and 1,4-anhydro-DL-ribitol. If acetonitrile is substituted for hexa-methylphosphoric triamide, the cyclic sulfites are the main products, and only traces of the chlorinated sugars are formed. ¹H- and ¹³C-n.m.r.-spectral analysis of these reactions demonstrated that one of the diastereomers preponderates. The structure of these cyclic sulfites was established by comparison of the ¹H-n.m.r. spectra with those of the propylene sulfites. Treatment of 1,2-O-isopropylidene-α-D-glucofuranose (14) with thionyl chloride in hexamethylphosphoric triamide yields 3-chloro-3-deoxy-1,2-O-isopropylidene-α-D-allofuranose 5,6-cyclic suffite. In contrast to the 2,3-cyclic suffites, which are stable, the cyclic sulfites derived from 14 slowly decompose at room temperature.     

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.     

2,4-Dinitrophenylhydrazides of polysialogangliosides

Treatment with 2,4-dinitrophenylhydrazine HCl in the presence of dicyclohexylcarbodiimide, converts gangliosides to their dinitrophenylhydrazides. This derivatization is the basis of a useful method for HPLC determination of gangliosides (K. Miyazaki, N. Okamura, Y. Kishimoto and Y. C. Lee (1986) Biochem. J. 235, 755-761). This procedure, however, yields two different GT1b products. By characterizing these two products using plasma desorption mass spectrometry, proton magnetic resonance and other chemical and physical techniques, we found that either one or two of the three sialic acid carboxyl groups in GT1b, were converted to dinitrophenylhydrazides. The remaining underivatized carboxyl groups formed lactones with hydroxyl groups from other carbohydrate residues. Also, while sialic acid residues of GD1a were fully derivatized, only one sialic acid in GD1b, two sialic acids in GT1a and two in GQ1b were converted to dinitrophenylhydrazides, the remaining carboxyl groups probably forming lactones. Sialic acid residues between galactose of the gangliotetraose chain and another sialic acid in polysialogangliosides appear to be underivatized possibly because of steric hindrance.     

Positive-negative, chemical-ionization, direct exposure mass spectrometry of underivatized prostaglandins

Nine underivatized prostaglandins were examined using direct exposure, ammonia, chemical-ionization, pulsed positive-negative ion mass spectrometry. The positive ion spectra were characterized by (M+18)+ ion adducts. The negative ion spectra were characterized by ions which depended upon the functionality present in the cyclopentane ring system (acetal for TXB2). The E and D series prostaglandins gave (M-18)- as the major negative ion, while the F series and TXB2 were characterized by negative ions corresponding to (M-1)-, and PGA2 by the parent (M)- ion. Prostaglandin 6-keto-PGF1 alpha was anomalous in this respect showing apparent dehydration, interpreted as an overall (M-18+1)+ and (M-18-1)- in the positive and negative ion spectra, respectively. All major ion types were shown to give essentially a linear response with respect to concentration in the 10-1000 ng range. Although these initial studies were conducted under ideal conditions, it would appear that direct chemical ionization techniques show promise for providing direct structural information on prostaglandins without the need for prior chemical derivatization.     

One-step fractionation of neutral and acidic glycosphingolipids by high-performance liquid chromatography

A procedure for a simultaneous separation of ganglioside components and neutral glycolipid components by high-performance liquid chromatography was described. One column packed with DEAE-derivatized controlled-pore glass (DEAE-CPG) was serially connected to two columns of underivatized, controlled-pore glass (CPG). A mixture of gangliosides and neutral glycolipids were loaded on DEAE-CPG and eluted with a mixture of chloroform-methanol-water, with increasing methanol and water (the first-phase gradient elution), followed by elution with increasing concentrations lithium acetate from 0.015 to 0.1 M in a mixture of chloroform-methanol-water (the second-phase gradient elution). Neutral glycolipids, mono- to hexaglycosylceramides , were separated within 80 min of the first-phase gradient elution, and mono- to tetrasialosylgangliosides were separated during the second-phase gradient elution within 60 min. The method has been applied to the determination of glycolipids isolated from rat tissues, and the procedure was found to be highly reproducible.     

Aberrant fatty acyl alpha-hydroxylation in human neuroblastoma tumor gangliosides

Abnormalities of ganglioside structure characterize the neoplastic state, and aberrant glycosylation has been implicated as underlying many new tumor ganglioside structures. However, variations in ceramide structure can also result in novel tumor gangliosides. To address systematically this aspect of ganglioside metabolism, we have initiated a study of the structures of the ceramide species of an oligosaccharide-homogeneous human tumor-derived ganglioside, GM2. The ganglioside was isolated from neuroblastoma tissue and purified by normal-phase high pressure liquid chromatography. Marked ceramide heterogeneity was observed; 18 individual ceramide species of neuroblastoma GM2 were separated by reversed-phase high pressure liquid chromatography and collected. Their structures were determined by a combination of negative- and positive-ion fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry of the underivatized gangliosides. The striking finding was the detection of alpha-hydroxylation of a significant fraction of each of the major fatty acid species (16:0, 18:0, 20:0, 22:0, and 24:1); alpha-hydroxylated species quantitatively represented almost one-fifth of the total tumor GM2 species. Fatty acyl hydroxylation was also detected in the ceramide of several other human tumor gangliosides. In contrast, as previously known, fatty acyl hydroxylation was not detected in the normal human brain gangliosides GM3, GM2, and GM1. We propose that aberrant fatty acid alpha-hydroxylation is a novel and sometimes quantitatively significant characteristic of human tumor ganglioside metabolism.     

Cord-factor analogs: synthesis of 6,6'-di-O-mycoloyl- and -corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside)

Appropriate solvolysis of 2,3,2',3'-tetra-O-benzyl-4,6,4', 6'-tetra-O-mesyl-alpha,alpha-trehalose gave 2,3,2',3' -tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (2). Selective tosylation or mesylation of 2 respectively gave the 6, 6'-ditosylate (3) and 6,6'-dimesylate (4), the structures of which were confirmed by the 1H-n.m.r. spectra of the corresponding 4,4'-di-O-acetyl derivatives. Treatment of 3 with potassium mycolate in toluene, and subsequent hydrogenolysis, gave the 6'-mycolate 6-tosylate derivative. Treatment of 3 with potassium mycolate or potassium corynomycolate in hexamethylphosphoric triamide, followed by catalytic hydrogenolysis, yielded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) and 6,6'-di-O-corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside). The same 6,6'-diesters were obtained from the 6,6'-dimesylate 4. Putative 4,6-anhydro-6'-monomycolates are also described.     

Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

Synthesis of benzyl and methyl 3-benzamido-2,3,6-trideoxy-2-fluoro-β-l-galactopyranoside: Protected C-2 fluoro analogues of daunosamine

The reaction of benzyl 2-benzamido-4,6-O-benzylidene-2-deoxy-3-O-tosyl-α-d-glucopyranoside or benzyl 4,6-O-benzylidene-2,3-benzoylepimino-2,3-dideoxy-α-d-allopyranoside with anhydrous tetrabutylammonium fluoride in hexamethylphosphoric triamide gave ∼40% of benzyl 3-benzamido-4,6-O-benzylidene-2,3-dideoxy-2-fluoro-α-d-altropyranoside (6a). Transformation of 6a into benzyl 3-benzamido-2,3,6-trideoxy-2-fluoro-α-d-arabino-hex-5-enopyranoside (13a) was carried out by well-established methodology. Hydrogenation of the double bond in 13a furnished the title compound in good yield. Methyl 3-benzamido-2,3,6-trideoxy-2-fluoro-β-l-galactopyranoside was also prepared in nine steps from 2-amino-2-deoxy-d-glucose.     

A method for profiling gangliosides in animal tissues using electrospray ionization-tandem mass spectrometry

Gangliosides are critical in many functions of mammalian cells but present as a minor lipid component with many molecular species of subtle differences. Conventional strategies for profiling gangliosides suffer from poor reproducibility, low sensitivity, and low-throughput capacity. Prior separation of gangliosides by thin-layer chromatography and/or high-performance liquid chromatography not only was laborious and tedious but also could introduce uneven losses of molecular species. We developed a new strategy of using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to profile gangliosides with high-throughput potential. This strategy involves three new findings: (i) collision-induced fragmentation of gangliosides gave rise to a common ion of m/z 290, a derivative of N-acetylneuraminic acid; (ii) phospholipids exert a profound suppression of ganglioside detection in ESI-MS/MS to prevent a direct detection in total cellular lipid extracts; and (iii) enrichment of gangliosides in the aqueous phase from total cellular lipid extracts eliminates the damping effect of phospholipids and permits direct precursor scan.