Mass Spectrometric Analysis of Oligo- and Polysialic Acids.

Oligo- and polysialic acids (oligo/polySia) are involved in numerous biological processes depending on the chain length, the comprised type of sialic acids, as well as the glycosidic linkages. Here, we describe the determination of the composition, the sequence, as well as the linkages between the sialic acid residues of lactonized oligo/polySia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)(/MS) and electrospray-ionization (ESI)-MS((n)).

Annotation of glycomics MS and MS/MS spectra using the GlycoWorkbench software tool.

The GlycoWorkbench software tool allows users to semiautomatically annotate glycomics MS and MS/MS spectra and MS glycoproteomics spectra. The GlycanBuilder software tool is embedded within GlycoWorkbench allowing users to draw glycan structures and export images of the drawn structures. This chapter demonstrates to users how to draw glycan structures within GlycoWorkbench using the GlycanBuilder software tool. This chapter also demonstrates how to use GlycoWorkbench to import MS and MS/MS glycomics spectra and use the cascading annotation feature to annotate both the MS and MS/MS spectra with a single command.

The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments.

During the EUROCarbDB project our group developed the GlycanBuilder and GlycoWorkbench glycoinformatics tools. This short communication summarizes the capabilities of these two tools and updates which have been made since the original publications in 2007 and 2008. GlycanBuilder is a tool that allows for the fast and intuitive drawing of glycan structures; this tool can be used standalone, embedded in web pages and can also be integrated into other programs. GlycoWorkbench has been designed to semi-automatically annotate glycomics data. This tool can be used to annotate mass spectrometry (MS) and MS/MS spectra of free oligosaccharides, N and O-linked glycans, GAGs (glycosaminoglycans) and glycolipids, as well as MS spectra of glycoproteins.

Elucidation of a novel lacto-N-decaose core structure in human milk using nonlinear analytical technique combinations.

Detailed structural analysis of high molecular weight human milk oligosaccharides (HMOs) is still a challenging task. Here we present a modular strategy for a flexible de novo structural characterization of this class of molecules. The protocol combines established techniques such as separation by two-dimensional high-performance liquid chromatography with different types of mass spectrometry, exoglycosidase digestion, and linkage analysis in an individual glycan-based manner. As a proof of principle, this approach was applied to two distinct HMO isomers representing a difucosylated octaose core and a trifucosylated decaose core. Obtained data revealed the presence of one terminal Lewis A and one internal Lewis X epitope in the case of the octaose and led to the identification of this molecule as a difucosylated iso-lacto-N-octaose. The trifucosylated, doubly branched lacto-N-neo-decaose was shown to represent a new type of HMO core structure in which the branched antenna is linked to carbon atom 3 of the innermost galactosyl residue. Hence, using this analytical protocol a novel HMO structure could be defined. Our results further demonstrate that a combination of different techniques may be required for de novo structural analysis of these molecules.

High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides.

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 µL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex(2)Fuc(2) and Hex(3)HexNAc(1)Fuc(2), as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a-b+), Le(a+b-), and Le(a-b-), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks.

EUROCarbDB: An open-access platform for glycoinformatics.

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.

Mass spectrometric fragmentation analysis of oligosialic and polysialic acids.

Oligosialic and polysialic acids (oligo/polySia) are characterized by high structural diversity, because of different types of sialic acids and glycosidic linkages. Although several methods have been described for the analysis of oligo/polySia, only high-performance liquid chromatography (HPLC) analysis in conjunction with 1,2-diamino-4,5-methylenedioxybenzene labeling, fluorometric C7/C9 detection, Western blotting, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS) of lactonized oligo/polySia species, require submicrogram amounts of analyte. Since these methods do not provide detailed structural information, this study is focused on the characterization of oligo/polySia by tandem mass spectrometry (MS/MS). MALDI-TOF-MS/MS and electrospray ionization tandem mass spectrometry (ESI-MS/MS), employing up to three cycles of ion isolation and fragmentation in an ion trap, have been used for the characterization of nonderivatized glycans, oligoSia species modified at their reducing or nonreducing ends, as well as partially O-acetylated oligoSia derivatives. The obtained spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. However, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-chained oligoSia and long polySia species. Furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues.

Characterization of the acidic N-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach.

Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5-2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-beta-galactosidase. Our results verified the presence of high-mannose-type Man(5)GlcNAc(2) compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.

GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.

Mass spectrometry is the main analytical technique currently used to address the challenges of glycomics as it offers unrivalled levels of sensitivity and the ability to handle complex mixtures of different glycan variations. Determination of glycan structures from analysis of MS data is a major bottleneck in high-throughput glycomics projects, and robust solutions to this problem are of critical importance. However, all the approaches currently available have inherent restrictions to the type of glycans they can identify, and none of them have proved to be a definitive tool for glycomics. GlycoWorkbench is a software tool developed by the EUROCarbDB initiative to assist the manual interpretation of MS data. The main task of GlycoWorkbench is to evaluate a set of structures proposed by the user by matching the corresponding theoretical list of fragment masses against the list of peaks derived from the spectrum. The tool provides an easy to use graphical interface, a comprehensive and increasing set of structural constituents, an exhaustive collection of fragmentation types, and a broad list of annotation options. The aim of GlycoWorkbench is to offer complete support for the routine interpretation of MS data. The software is available for download from: http://www.eurocarbdb.org/applications/ms-tools.

"Glyco-peakfinder"--de novo composition analysis of glycoconjugates.

Mass spectrometric techniques are the key technology for rapid and reliable glycan analysis. However, the lack of robust, dependable, and freely available software for the (semi-) automatic annotation of mass spectra is still a severe bottleneck that hampers their rapid interpretation. In this article the "Glyco-Peakfinder" web-service is described allowing de novo determination of glycan compositions from their mass signals. Starting from a basic set of mandatory masses of glycan components, the calculation can be performed without any knowledge concerning the biological background of the sample or the fragmentation technique used. "Glyco-Peakfinder" assigns all types of fragment ions including monosaccharide cross-ring cleavage products and multiply charged ions. It provides full user control to handle modified glycans (persubstituted molecules, reducing-end modifications, glycoconjugates) and ion types. The formula applied to calculate the fragment masses and an outline of the implemented algorithm are discussed. A systematic evaluation of the dependence of all factors influencing the computation time revealed strikingly different impact of the individual calculation steps. To provide access to known carbohydrate structures a "composition search" in the open access database GLYCOSCIENCES.de can be performed. The service is available at the URL: www.eurocarbdb.org/applications/ms-tools.

Structural characterization of N-glycans from the freshwater snail Biomphalaria glabrata cross-reacting with Schistosoma mansoni glycoconjugates.

The human parasitic trematode Schistosoma mansoni has a complex life cycle that includes the freshwater snail Biomphalaria glabrata as intermediate host. Within each stage, the parasite synthesizes a wide array of glycoconjugates, exhibiting, in part, unique carbohydrate structures. In addition, the parasite expresses definitive host-like sugar epitopes, such as Lewis X determinants, supporting the concept of carbohydrate-mediated molecular mimicry as an invasion and survival strategy. In the present study, we investigated whether common carbohydrate determinants occur also at the level of the intermediate host. To this end, a structural characterization of hemolymph glycoprotein-N-glycans of B. glabrata was performed. N-glycans were released from tryptic glycopeptides and labeled with 2-aminopyridine. Sugar chains serologically cross-reacting with S. mansoni glycoconjugates were isolated by immunoaffinity chromatography using a polyclonal antiserum directed against schistosomal egg antigens and fractionated by Aleuria aurantia lectin affinity chromatography and high-performance liquid chromatography. Obtained glycans were analyzed by different mass spectrometric techniques as well as by monosaccharide constituent and linkage analysis. The results revealed a highly heterogeneous oligosaccharide pattern. Cross-reacting species represented about 5% of the total glycans and exhibited a terminal Fuc(alpha1-3)GalNAc unit, a (1-2)-linked xylosyl residue, or both types of structural motifs. In conclusion, our study demonstrates the presence of common carbohydrate epitopes also at the level of S. mansoni and its intermediate host.