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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.

Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (M[S.sup.n]) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following M[S.sup.2] and required multiple (M[S.sup.n>2]) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation 'scars' throughout the M[S.sup.n] tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the M[S.sup.n] data sets.

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 M[S.sup.n] 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 M[S.sup.n] 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.

A bottom-up approach to achieve full oligosaccharide and glycan characterization has been described that is based on an M[S.sup.n] fragment spectral library and associated tools. The library, identified as FragLib, was initiated with known standards and commercially available oligomers prepared as methylated derivatives. As a component of this effort a set of software tools has been written for storing, organizing, and comparing spectral files, including the identification of isobaric mixtures. These tools provide a facile and objective evaluation of structural details including inter-residue linkage, monomer identification, anomeric configuration, and branching. The tools are components of a web-based data sharing interface for sample tracking, spectral searching, and structural confirmation. Applications have been detailed with unknown samples and previously characterized glycoconjugates.

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 (M[S.sup.n]) 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 MS' 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 raptures 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 human CD1b protein presents lipid antigens to T cells, but the molecular mechanism is unknown. Identification of mycobacterial glucose monomycolate (GMM) as a CD1b-presented glycolipid allowed determination of the structural requirements for its recognition by T cells. Presentation of GMM to CD1b-restricted T cells was not affected by substantial variations in its lipid tails, but was extremely sensitive to chemical alterations in its carbohydrate or other polar substituents. These findings support the view that the recently demonstrated hydrophobic CD1 groove binds the acyl chains of lipid antigens relatively nonspecifically, thereby positioning the hydrophilic components for highly specific interactions with T cell antigen receptors., Human CD1 proteins are a family of nonpolymorphic transmembrane glycoproteins expressed in association with [Β.sub.2]-microglobulin on the surface of antigen-presenting cells (APCs) (1, 2). Unlike antigen-presenting molecules encoded in the [...]

E-selectin binding gangliosides from myelogenous leukemia HL60 cells were studied to examine their E-selectin binding pattern and to compare it with that of human neutrophils. Both E-selectin nonbinding and nonbinding fractions were analyzed using a variety of techniques. The major gangliosides present in each fraction are presented. The characteristic structures of the gangliosides are also provided.

Gangliosides extracted from myelogenous leukemia HL60 cells and human leukocytes were extensively fractionated and analyzed to examine the E-selectin binding ability of the respective fractions. High performance thin layer chromatography revealed that only monosialogangliosides having a polylactosamine core with less than 10 monosaccharide units exhibited E-selectin binding. None of the fractions exhibited P-selectin binding. The structures of the major gangliosides of HL60 cells are provided.

Structure of exopolysaccharide succinoglycan of Rhizobium meliloti Rm1021 is studied by collision-induced dissociation of octameric oligosaccharide repeating unit and electrospray ionization. Residue positions of the acetyl and succinyl modified octamer repeating complex are identified. Glycosidic linkages and pyranose ring positions of succinyl and acetyl modifications are detected.

Glycosaminoglycans (GAGs) are carbohydrate polyionic polymers that participate in a host of critically important biological processes. A significant difficulty in the comprehensive structural characterization of GAGs is the determination of specific sulfation position isomers. We chose to circumvent sulfate lability by its liberation followed by specific isotope exchange that makes it amenable to methylation, collisional induced dissociation, and MSn disassembly for a detailed structural characterization. A set of chemistries that include sulfate release, isotopic (CD3– and CD3–CO–) replacement, and methylation have been modified to yield a stable product ideal for sequencing by MSn. Disassembly of these samples provides a detailed read-out of sequence inclusive of all sulfation sites. As documenting steps, we applied these chemical modifications to a series of disaccharides and a synthetic GAG pentamer, Arixtra®. Upon disassembly, glycosidic and cross-ring cleavages define the monomer composition including individual sulfation positions. The N- and O-sulfates are differentiated by deuterium-containing mass compositions. The uronic methylesters do not significantly alter the fragmentation patterns. A fragment library of these products is being assembled as an adjunct to our larger fragment library, some 15 years in the making. [ABSTRACT FROM AUTHOR]