This report is the first in a series of three focused on establishing congruent strategies for carbohydrate sequencing. The reports are divided into (i) analytical considerations that account for all aspects of small oligomer structure by MS n disassembly, (ii) database support using an ion fragment library and associated tools for highthroughput analysis, and (iii) a concluding algorithm for defining oligosaccharide topology from MS n disassembly pathways. The analytical contribution of this first report explores the limits of structural detail exposed by ion trap mass spectrometry with samples prepared as methyl derivatives and analyzed as metal ion adducts. This data mining effort focuses on correlating the fragments of small oligomers to stereospecific glycan structures, an outcome attributed to a combination of metal ion adduction and analyte conformation. Facile glycosidic cleavage introduces a point of lability (pyranosyl-1-ene) that upon collisional activation initiates subsequent ring fragmentation. Product masses and ion intensities vary with interresidue linkage, branching position, and monomer stereochemistry. Excessive fragmentation is the property of small oligomers where collisional energy within a smaller number of oscillators dissipates through extensive fragmentation. The procedures discussed in this report are unified into a singular strategy using an ion trap mass spectrometer with the sensitivity expected for electron multiplier detection. Although a small set of structures have been discussed, the basic principles considered are fully congruent, with ample opportunities for expansion.The proliferation of reports attributing biological function to oligosaccaharide epitopes continues unabated. To fully appreciate their specific biological roles, an improved accounting of carbohydrate structural details needs greater scrutiny and more judicious reporting. Unfortunately, a comprehensive strategy for carbohydrate sequencing is lacking. Over the past decades, the absence of an integrated strategy to fully define a carbohydrate sequence has been given little attention due to overwhelming and direct functional lineage of nucleic acid and protein polymers. Even selective strategies to assign components of structure show little focus toward congruency. The reporting of partial sequences, assumed motifs, sequence arrays devoid of linkage and branching definition, and multiple structures enclosed in brackets are currently acceptable conclusions in published reports. The general acceptance of such reports diminishes the driving force for the development of improved and comprehensive methodologies.A sequence definition should provide all the components of structure necessary for reporting or synthesis. Structural components should include monomer identification, positions of interresidue linkage, and array topology, where a linkage definition includes anomericity, and array topologies describe linear and branching sequence information. When considering molecular glycosylation, sites of conjugatio...

Human antibody 2G12 broadly neutralizes human immunodeficiency virus type 1 (HIV-1) isolates and shows protective activity against viral challenge in animal models. Previous mutational analysis suggested that 2G12 recognized a novel cluster of high-mannose type oligosaccharides on HIV-1 gp120. To explore the carbohydrate antigen for HIV-1 vaccine design, we have studied the binding of 2G12 to an array of HIV-1 high-mannose type oligosaccharides by competitive ELISAs and found that Man9GlcNAc is 210- and 74-fold more effective than Man5GlcNAc and Man6GlcNAc in binding to 2G12. The results establish that the larger high-mannose oligosaccharide on HIV-1 is the favorable subunit for 2G12 recognition. To mimic the putative epitope of 2G12, we have created scaffold-based multivalent Man9 clusters and found that the galactose-scaffolded bi-, tri-, and tetra-valent Man9 clusters are 7-, 22-, and 73-fold more effective in binding to 2G12 than the monomeric Man9GlcNAc2Asn. The experimental data shed light on further structural optimization of epitope mimics for developing a carbohydrate-based HIV-1 vaccine.

A bottom-up approach to achieve full oligosaccharide and glycan characterization has been described that is based on an MS 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 interresidue 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.The preceding report in this series presented data indicating that spectra from various stages of ion trap disassembly (MS n ) could supply structural details for the characterization of linkage, monomer ID, and branching. 1 With complete analysis as a practical consideration, there is a fundamental need for high-throughput tools to complement proteome studies. In that regard, little could be more important than searchable spectral library files for structural confirmation. The general reproducibility of ion trap (IT) spectra, and energy independence from the modes of ionization and collisional activation (CA), make compiling an MS n library an important research consideration.A number of oligosaccharide libraries have been reported each having specific characteristics applicable to the needs of independent research groups. GlycosidIQ 2 was developed for computerized interpretation of oligosaccharide spectra based on matching experimental data with theoretical fragments generated from GlycoSuiteDB, 3,4 a database of glycan structures reported in the literature. Similarly, the GlycoFragment/GlycoSearchMS package compares each peak of an observed mass spectrum with the calculated fragments of all structures contained in the SweetDB database to support the interpretation of mass spectra of complex carbohydrates. 5 Both GlycosidIQ and GlycoFragment/GlycoSearchMS utilize tandem MS (MS 2 ) of native samples and theoretically generated spectra as reference. Tseng et al. applied a "catalog library" approach for elucidating the structures of minor components in a mixture of oligosaccharides. 6 The catalog data consist of the characteristic fragmentation patterns belonging to a set of specific substructures compiled from a library of known compounds present in the same sample that have also been characterized by other techniques. Collisioninduced disassociation (CID) was used to determine the presence of the cataloged motifs. Recently, a standard trisaccharide MS 2 library has been reported that uses underivatized synthetic samples. 7 * Corresponding author. Tel: (603) In this report, we summarize initial progress and introduce simple ...

Background Group B streptococcus (GBS) causes serious diseases in newborn infants, often resulting in lifelong neurologic impairments or death. Prophylactic vaccination of pregnant women prior to delivery could provide comprehensive protection, as early onset and late-onset disease and maternal complications potentially could be addressed. Methods Capsular polysaccharide conjugate vaccine GBS6 was designed using surveillance data yielded by whole-genome sequencing of a global collection of recently recovered GBS isolates responsible for invasive neonatal GBS disease. Capsular polysaccharides were isolated, oxidized using sodium periodate, and conjugated to CRM 197 by reductive amination in dimethyl sulfoxide. Immune responses in mice and rhesus macaques were measured in a multiplex Luminex immunoglobulin G (IgG) assay and opsonophagocytic activity assays. Results The optimized conjugates were immunogenic, alone and in combination, in mice and rhesus macaques, inducing IgG antibodies that mediated opsonophagocytic killing. Active immunization of murine dams with GBS6 prior to mating resulted in serotype-specific protection of pups from a lethal challenge with GBS. Protection following passive administration of serotype-specific IgG monoclonal antibodies to dams demonstrated conclusively that anticapsular polysaccharide IgG alone is sufficient for protection. Conclusions The findings support the ongoing clinical evaluation of maternal GBS6 vaccination as a potential alternative method to prevent GBS disease in infants.