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Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization--time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-D-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its beta-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) beta-L-rhamnose (1-3) beta-N-acetyl-D-glucosamine (1-] with an N-acetyl-D-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

Thirteen high mannose isomers have been structurally characterized within three glycomers, Man(5)GlcNAc(2), Man(7)GlcNAc(2), and Man(8)GlcNAc(2) released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. Three previously characterized isomers from Man(7) and Man(8) (three each) have been identified plus one unreported Man(7) isomer. Incomplete alpha-glucosidase activity on the Man(6) and Man(7) glycoproteins appears to account for two additional isomeric structures. The preeminence of ion traps for detail analysis was further demonstrated by resolving three new isomers within the Man(5) glycomer summing to the six previously unreported structures in this glycoprotein. All reported structures represent a distribution of Golgi processing remnants that fall within the Man(9)GlcNAc(2) footprint. Topologies were defined by ion compositions along a disassembly pathway while linkage and branching were aided by spectral identity in a small oligomer fragment library. Isomers from this glycoprotein appear to represent a distribution of Golgi processing remnants, and an alphanumeric classification scheme has been devised to identify all products. Although numerous analytical strategies have been introduced to identify selected components of structure, it has been the continued focus of this and previous reports to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Duplication of these and results from comparable standards could bring an important analytical focus to carbohydrate sequencing that is greatly lacking.

This is the third in a sequence of reports devoted to the development of congruent strategies for carbohydrate sequencing. Two previous reports outlined the strategies for observing structural detail from MSn data and introduced tools that compile, search, and compare fragment spectra in a bottom-up approach to oligosaccharide sequencing. In this third report, we introduce the operational details of an algorithm that we define as the Oligosaccharide Subtree Constraint Algorithm (OSCAR). This algorithm assimilates analyst-selected MSn ion fragmentation pathways into oligosaccharide topology (branching and linkage) using what may be considered a top-down sequencing strategy. Guided by a series of logical constraints, this de novo algorithm provides molecular topology without presumed biosynthetic constraints or external comparisons. In this introductory study, OSCAR is applied to a series of permethylated oligomers and isomeric glycans, and topologies are assigned in a few hundredths of a second.

Several oligosaccharide and N-linked glycan samples have been utilized to evaluate structural detail obtained with an ion trap mass spectrometer (ITMS). Using multistage MS/MS (MSn) in a commercial instrument, linear sequence, linkage, and branching have been studied, as well as the positional isomers within branched glycans. Samples were prepared as methylated derivatives, which amplifies the structurally informative cross-ring and double glycosidic cleavage products associated with interresidue linkage and oligomer branching detail. Spectral analysis of a linear glucose homopolymer demonstrated ITMS to be comparable with data obtained from a triple-quadrupole instrument, and in a study of complex glycans, the feature of MSn enabled the assignment of linkages that cannot be assessed by conventional triple-quadrupole tandem instrumentation. The longer time scale of CID experiments enhances the abundance of multiple bond ruptures important for a complete understanding of structure. This technology appears to provide a significant step toward the goal of characterizing all aspects of carbohydrate structure using a single instrument.

The improved sensitivity and soft ionization characteristics of electrospray (ES) mass spectrometry (MS) has been applied to the glycan structures of recombinant erythropoietin (rEPO). The four glycopeptides (O-126, N-24, N-38, N-83) were mapped, isolated, deglycosylated, and the glycans profiled (without desialylation) by ES-MS as their methyl derivatives. The O-linked glycopeptide was also analyzed directly. In this fraction seven glycans were identified (two previously unreported) in addition to the unglycosylated peptide. The three N-linked fractions were divided with one fraction preceded by periodate oxidation and reduction to resolve isomeric structures, linkage, and branching patterns, and confirm the overall structural motif. All were methylated and profiled. Two biantennary, 8 triantennary, and 10 tetraantennary structures were identified with approximately 13% of each N-linked glycan possessing a single N-glycolylneuraminyl analog. The minimal energies of ionization, the absence of a matrix background, and enhanced sensitivity brings an improved technology for studying carbohydrate structural detail.

The detailed structure of the symbiotically important exopolysaccharide succinoglycan from Rhizobium meliloti Rm1021 was determined by mass spectrometry with electrospray ionization and collision-induced dissociation of the octameric oligosaccharide repeating unit. Previously undetermined locations of the succinyl and acetyl modifications were determined, in respect to both residue locations within the octamer and the carbon positions within the pyranose ring. Glycosidic linkages determined previously by methylation analysis were also verified.

A Golgi preparation from Spodoptera frugiperda (IPLB-SF-21AE) cells was incubated in the presence of the mannosidase II inhibitor, swainsonine, with the oligosaccharide, M(alpha 1,3)[[M(alpha 1,3)[M(alpha 1,6)]M(alpha 1,6)]] M(beta 1,4)Gn(beta 1,4)Gn (M5Gn2), the preferred substrate for the enzyme, UDP-N-acetylglucosamine:alpha-3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (Gn-TI). This resulted in formation of the product, Gn(beta 1,2)M(alpha 1,3)[[M(alpha 1,3)[M(alpha 1,6)]M(alpha 1,6)]]- M(beta 1,4) Gn(beta 1,4)Gn (Gn(I)M5Gn2). A significantly increased (> 4-fold) rate of conversion of M5Gn2 to Gn(I)M5Gn2 occurred with insect cell-derived Golgi preparations that had been infected with a recombinant baculovirus for 66 h, a time at which significant amounts of complex-type oligosaccharides were assembled on a heterologous protein, human plasminogen, expressed in this system. Coupled with previous results (Davidson, D.J., Bretthauer, R.K., and Castellino, F.J. (1991) Biochemistry 30, 9811-9815) that demonstrated the occurrence of virally induced activation of a specific M6-mannosidase in IPLB-SF-21AE cells, it is clear that viral infection of lepidopteran insect cells makes available enzymes that provide and utilize the substrate, M5Gn2-protein. This is a key feature in the explanation of the previous original observations made by this laboratory, that lepidopteran insect cells are capable of assembly of complex-type oligosaccharides on glycoproteins.

In Rhizobium leguminosarum biovar viciae, the nodABC and nodFEL operons are involved in the production of lipo-oligosaccharide signals which mediate host specificity. The structure of these metabolites and those produced in nod mutants links the nodE and nodL genes to specific chemical features of the signal molecules. A nodE-determined, highly unsaturated fatty acid and a nodL-determined O-acetyl substituent are essential for the ability of the signals to induce nodule meristems on the host plant Vicia sativa.

N-Linked glycans have been characterized by supercritical fluid chromatography (SFC) and SFC-MS using positive- and negative-ion chemical ionization. Four common oligosaccharide derivatives have been prepared and their chromatographic properties assessed on three SFC columns of varying polarity. Carbon dioxide has been used as the SFC mobile phase, with ammonia or CO2 added to the ion source for positive- and negative-ion chemical ionization, respectively. Direct SFC-MS interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Positive ammonia chemical ionization yields abundant molecular-weight information, (MH)+, and (MNH4)+ with little or no fragmentation. To capitalize on sensitivity, samples were prepared with the pentafluorobenzyl aminobenzoate reagent, acetylated, and analyzed by SFC-NICI-MS. This modification improves column efficiency and resolution and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.

A bimodal reagent (pentafluorobenzyl aminobenzoate) has been synthesized to improve oligosaccharide isolation, detection, and structural characterization. The reagent is glycosidically attached to the reducing end of glycan residues, imparts fluorescent and uv properties for chromatographic detection, and functions as an efficient electron trap under negative ion chemical ionization mass spectrometry for femtomole detectability. Facile ester cleavage and pentafluorobenzyl elimination provides a single molecular-weight-related fragment in high abundance. Procedures are described for reagent synthesis, purification, and oligosaccharide conjugation. Carbohydrate samples derivatized with this reagent are evaluated by high-performance liquid chromatography and supercritical fluid chromatography (SFC) and for sensitivity by SFC negative ion chemical ionization mass spectrometry.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.

Lysosomal enzymes isolated from the slime mold Dictyostelium discoideum bind to the mannose 6-phosphate receptor which is present in many mammalian cells. While binding to the receptor suggests that the slime mold enzymes possess the same mannose 6-phosphate recognition marker as their mammalian counterparts, initial structural studies of the phosphorylated oligosaccharides have indicated that the phosphate is attached to high mannose-type units via an unusual phosphodiester linkage (Freeze, H.H., Yeh, R., Miller, A.L., and Kornfeld, S. (1983) J. Biol. Chem. 258, 14874-14879). To identify the components of the phosphodiester group we have isolated the phosphorylated high-mannose oligosaccharides from D. discoideum AX3 cells labeled with [2-3H]mannose or [6-3H]glucosamine and from the differentiation medium of unlabeled cells. The major phosphorylated species contain one or two phosphodiester groups and an average of 6 or 7 mannose residues. The phosphodiesters are relatively stable to both acid and base hydrolysis, but upon strong acid hydrolysis (conditions that completely hydrolyze the oligosaccharide) mannose 6-phosphate residues are liberated. Through a combination of techniques, including fast atom bombardment and direct chemical ionization mass spectrometry, it is shown that the mannose 6-phosphate residues of the intact oligosaccharide are diesterified to methyl groups. This indicates that slime mold possesses a different biosynthetic pathway for the formation of phosphorylated high mannose-type oligosaccharides than is utilized by higher organisms.

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.

Fast atom bombardment (FAB) ionization and two coupled analyzers (BE-EB) have been combined with neutral gas collision (C) to enhance structural information in the mass spectra of oligosaccharides. (B and E are abbreviations for magnetic and electric sectors respectively.) FAB ionization and the first analysers (BE) have provided parent ions free from biological and liquid matrix contaminants. Structural detail of these products were observed after collision and daughter ion analysis in a second coupled analyser (EB). Starting from complex mixtures, this instrumental approach, BE-C-EB, has provided specific oligomeric sequence information which was not observed in the normal FAB mass spectra. Collision spectra obtained from isomeric linear and branched oligosaccharides show unique fragments that can be directly related to structure.

The cyclic (1----2)-beta-D-glucans produced by species of Agrobacterium and Rhizobium resemble the membrane-derived oligosaccharides of Escherichia coli in their periplasmic localization, intermediate size, and (1----2)-beta-D-glucan backbones. The regulation of the biosynthesis of cyclic (1----2)-beta-D-glucan by Agrobacterium tumefaciens is now shown to parallel the osmotic regulation of membrane-derived oligosaccharide biosynthesis in Escherichia coli. This result suggests a general role for periplasmic oligosaccharides in the osmotic adaptation of Gram-negative bacteria as ecologically diverse as enteric and soil bacteria.

We report herein the results of f.a.b.-m.s. experiments conducted on synthetic fragments of glycosaminoglycans, one of them representing the pentasaccharidic sequence present in heparin and responsible for the binding to antithrombin III, and the others being related to this sequence. The results indicate that f.a.b.-m.s. can be very useful for the structural analysis of sulfated glycosaminoglycans. The relatively small amounts of sample required enable molecular characterization at physiologically significant levels. In contrast to the chondroitin sulfates, the heparin saccharides analyzed and reported here do not provide sequence information. The data indicate that glycosidic rupture is not a process competing with the much more facile loss of N-sulfite residues. Dominating the spectra are a series of molecular-weight-related ions (distributed to indicate the associated countercation composition), and fragments related directly to sulfite elimination. This f.a.b.-induced, facile loss of sulfite may impose limitations in molecular-weight analysis for the larger oligomers.

To enhance resolution and detectability of carbohydrates by liquid chromatography, two fluorescent labels, introduced into oligosaccharides by reductive amination, were compared by use of standard sugars and a complex, biological sample of D-mannose oligomers obtained from the urine of a mannosidosis patient. Both labels, 2-aminopyridine and 7-amino-1-naphthol, improved the chromatographic efficiency and detection sensitivity. However, reductive amination with the pyridinylamine derivative was incomplete. The Schiff-base intermediates left in the mixtures were only partially resolved by chromatography and complicate the patterns. In contrast, the naphtholamine derivatives were completely reduced and, in addition, possess enhanced fluorescence.