Higher Energy Collisional Dissociation Mass Spectrometry of Sulfated O-Linked Oligosaccharides.

Sulfation is the final decoration of mucin-type O-linked oligosaccharides before mucins are released into the lumen of the gastrointestinal, respiratory, and genital tracts. Because only a fraction of oligosaccharides undergo this type of modifications in the Golgi apparatus, sometimes also only by dedicated cells, the glycobiology of these low abundant sulfated oligosaccharides is often overlooked. At the same time, the technology to consistently identify and characterize them has been lagging. We adopted higher energy collisional dissociation to characterize sulfated oligosaccharides from porcine gastric and human salivary MUC5B mucins. With this approach we could generate conclusive spectra up to nonasaccharides. Both singly and doubly sulfated oligosaccharides were characterized. By comparing the fragmentation of low-mass fragments of m/ z 100-320 with standards for six-linked and three-linked sulfate, it could be shown that characteristic fragmentation exists, verifying that porcine gastric mucin contains mostly six-linked sulfate to GlcNAc, whereas human MUC5B contains mostly three-linked Gal. When performing ion-trap MS2 fragmentation, these low-molecular-mass fragments are usually not detected. Hence it can be concluded that to be able to address biological questions of sulfation low-mass fragments are important for the assignment of sulfate position.

Validation of the curation pipeline of UniCarb-DB: building a global glycan reference MS/MS repository.

The UniCarb-DB database is an emerging public glycomics data repository, containing over 500 tandem mass spectra (as of March 2013) of glycans released from glycoproteins. A major challenge in glycomics research is to provide and maintain high-quality datasets that will offer the necessary diversity to support the development of accurate bioinformatics tools for data deposition and analysis. The role of UniCarb-DB, as an archival database, is to provide the glycomics community with open-access to a comprehensive LC MS/MS library of N- and O- linked glycans released from glycoproteins that have been annotated with glycosidic and cross-ring fragmentation ions, retention times, and associated experimental metadata descriptions. Here, we introduce the UniCarb-DB data submission pipeline and its practical application to construct a library of LC-MS/MS glycan standards that forms part of this database. In this context, an independent consortium of three laboratories was established to analyze the same 23 commercially available oligosaccharide standards, all by using graphitized carbon-liquid chromatography (LC) electrospray ionization (ESI) ion trap mass spectrometry in the negative ion mode. A dot product score was calculated for each spectrum in the three sets of data as a measure of the comparability that is necessary for use of such a collection in library-based spectral matching and glycan structural identification. The effects of charge state, de-isotoping and threshold levels on the quality of the input data are shown. The provision of well-characterized oligosaccharide fragmentation data provides the opportunity to identify determinants of specific glycan structures, and will contribute to the confidence level of algorithms that assign glycan structures to experimental MS/MS spectra. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Toolboxes for a standardised and systematic study of glycans.

BACKGROUND: Recent progress in method development for characterising the branched structures of complex carbohydrates has now enabled higher throughput technology. Automation of structure analysis then calls for software development since adding meaning to large data collections in reasonable time requires corresponding bioinformatics methods and tools. Current glycobioinformatics resources do cover information on the structure and function of glycans, their interaction with proteins or their enzymatic synthesis. However, this information is partial, scattered and often difficult to find to for non-glycobiologists. METHODS: Following our diagnosis of the causes of the slow development of glycobioinformatics, we review the "objective" difficulties encountered in defining adequate formats for representing complex entities and developing efficient analysis software. RESULTS: Various solutions already implemented and strategies defined to bridge glycobiology with different fields and integrate the heterogeneous glyco-related information are presented. CONCLUSIONS: Despite the initial stage of our integrative efforts, this paper highlights the rapid expansion of glycomics, the validity of existing resources and the bright future of glycobioinformatics.

Development and validation of the pediatric AzBio sentence lists.

OBJECTIVES: The goal of this study was to create and validate a new set of sentence lists that could be used to evaluate the speech-perception abilities of listeners with hearing loss in cases where adult materials are inappropriate due to difficulty level or content. The authors aimed to generate a large number of sentence lists with an equivalent level of difficulty for the evaluation of performance over time and across conditions. DESIGN: The original Pediatric AzBio sentence corpus included 450 sentences recorded from one female talker. All sentences included in the corpus were successfully repeated by kindergarten and first-grade students with normal hearing. The mean intelligibility of each sentence was estimated by processing each sentence through a cochlear implant simulation and calculating the mean percent correct score achieved by 15 normal-hearing listeners. After sorting sentences by mean percent correct scores, 320 sentences were assigned to 16 lists of equivalent difficulty. List equivalency was then validated by presenting all sentence lists, in a novel random order, to adults and children with hearing loss. A final-validation stage examined single-list comparisons from adult and pediatric listeners tested in research or clinical settings. RESULTS: The results of the simulation study allowed for the creation of 16 lists of 20 sentences. The average intelligibility of each list ranged from 78.4 to 78.7%. List equivalency was then validated, when the results of 16 adult cochlear implant users and 9 pediatric hearing aid and cochlear implant users revealed no significant differences across lists. The binomial distribution model was used to account for the inherent variability observed in the lists. This model was also used to generate 95% confidence intervals for one and two list comparisons. A retrospective analysis of 361 instances from 78 adult cochlear implant users and 48 instances from 36 pediatric cochlear implant users revealed that the 95% confidence intervals derived from the model captured 94% of all responses (385 of 409). CONCLUSIONS: The cochlear implant simulation was shown to be an effective method for estimating the intelligibility of individual sentences for use in the evaluation of cochlear implant users. Furthermore, the method used for constructing equivalent sentence lists and estimating the inherent variability of the materials has also been validated. Thus, the AzBio Pediatric Sentence Lists are equivalent and appropriate for the assessment of speech-understanding abilities of children with hearing loss as well as adults for whom performance on AzBio sentences is near the floor.

UniCarb-DB: An MS/MS Experimental Glycomic Fragmentation Database.

Glycosylation is a unique posttranslational modification that dynamically shapes the surface of cells. Glycans attached to proteins or lipids in a cell or tissue are studied as a whole and collectively designated as a glycome. UniCarb-DB is a glycomic spectral library of tandem mass spectrometry (MS/MS) fragment data. The current version of the database consists of over 1500 entries and over 1000 unique structures. Each entry contains parent ion information with associated MS/MS spectra, metadata about the original publication, experimental conditions, and biological origin. Each structure is also associated with the GlyTouCan glycan structure repository allowing easy access to other glycomic resources. The database can be directly utilized by mass spectrometry (MS) experimentalists through the conversion of data generated by MS into structural information. Flexible online search tools along with a downloadable version of the database are easily incorporated in either commercial or open-access MS software. This chapter highlights UniCarb-DB online search tool to browse differences of isomeric structures between spectra, a peak matching search between user-generated MS/MS spectra and spectra stored in UniCarb-DB and more advanced MS tools for combined quantitative and qualitative glycomics.

Statistical analysis of glycosylation profiles to compare tissue type and inflammatory disease state.

MOTIVATION: Glycosylation is one of the most important post-translational modifications of proteins and explains some aspects of the diversification of higher organisms not explained by template-driven synthesis. For glycomics to mature as much as genomics and proteomics, the necessary tools need to be developed and tested. Liquid chromatography-mass spectrometry is one of the gold standards for oligosaccharide analysis and leads to large amounts of data, not easily interpreted manually. We present a study on the testing and validation of statistical analysis tools to aid the structural elucidation of these analyses as well as using the results to answer biologically relevant questions. RESULTS: We show the usefulness of data reduction and statistical analysis in the interpretation of complex glycosylation data. The reduction does not result in the loss of importance of the glycosylation information as shown by comparison of control and disease samples in two tissue types.

UniCarb-DB: a database resource for glycomic discovery.

UNLABELLED: Glycosylation is one of the most important post-translational modifications of proteins, known to be involved in pathogen recognition, innate immune response and protection of epithelial membranes. However, when compared to the tools and databases available for the processing of high-throughput proteomic data, the glycomic domain is severely lacking. While tools to assist the analysis of mass spectrometry (MS) and HPLC are continuously improving, there are few resources available to support liquid chromatography (LC)-MS/MS techniques for glycan structure profiling. Here, we present a platform for presenting oligosaccharide structures and fragment data characterized by LC-MS/MS strategies. The database is annotated with high-quality datasets and is designed to extend and reinforce those standards and ontologies developed by existing glycomics databases. AVAILABILITY: http://www.unicarb-db.org

UniCarbKB: putting the pieces together for glycomics research.

Despite the success of several international initiatives the glycosciences still lack a managed infrastructure that contributes to the advancement of research through the provision of comprehensive structural and experimental glycan data collections. UniCarbKB is an initiative that aims to promote the creation of an online information storage and search platform for glycomics and glycobiology research. The knowledgebase will offer a freely accessible and information-rich resource supported by querying interfaces, annotation technologies and the adoption of common standards to integrate structural, experimental and functional data. The UniCarbKB framework endeavors to support the growth of glycobioinformatics and the dissemination of knowledge through the provision of an open and unified portal to encourage the sharing of data. In order to achieve this, the framework is committed to the development of tools and procedures that support data annotation, and expanding interoperability through cross-referencing of existing databases. Database URL: http://www.unicarbkb.org.

Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems.

Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive ß-elimination. Using LC-MS and LC-MS(2) with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exemplified by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O-glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS (n) experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.

Structural Identification of O-Linked Oligosaccharides Using Exoglycosidases and MSn Together with UniCarb-DB Fragment Spectra Comparison.

The availability of specific exoglycosidases alongside a spectral library of O-linked oligosaccharide collision induced dissociation (CID) MS fragments, UniCarb-DB, provides a pathway to make the elucidation of O-linked oligosaccharides more efficient. Here, we advise an approach of exoglycosidase-digestion of O-linked oligosaccharide mixtures, for structures that do not provide confirmative spectra. The combination of specific exoglycosidase digestion and MS2 matching of the exoglycosidase products with structures from UniCarb-DB, allowed the assignment of unknown structures. This approach was illustrated by treating sialylated core 2 O-linked oligosaccharides, released from the human synovial glycoprotein (lubricin), with a α2-3 specific sialidase. This methodology demonstrated the exclusive 3 linked nature of the sialylation of core 2 oligosaccharides on lubricin. When specific exoglycosidases were not available, MS3 spectral matching using standards was used. This allowed the unusual 4-linked terminal GlcNAc epitope in a porcine stomach to be identified in the GlcNAc1-4Galb1-3(GlcNAcb1-6)GalNAcol structure, indicating the antibacterial epitope GlcNAca1-4. In total, 13 structures were identified using exoglycosidase and MSn, alongside UniCarb-DB fragment spectra comparison. UniCarb-DB could also be used to identify the specificity of unknown exoglycosidases in human saliva. Endogenous salivary exoglycosidase activity on mucin oligosaccharides could be monitored by comparing the generated tandem MS spectra with those present in UniCarb-DB, showing that oral exoglycosidases were dominated by sialidases with a higher activity towards 3-linked sialic acid rather than 6-linked sialic acid.

Glycomic work-flow for analysis of mucin O-linked oligosaccharides.

The high-throughput analysis of the glycosylation of high molecular weight proteins, such as mucins, has been the aim of glycomics initiatives for the last decade. Here, we present a work-flow for the efficient and reproducible analysis of reduced oligosaccharides from a typical mucin sample. This work-flow can be applied to any similar samples of oligosaccharides. We include recently developed bioinformatic procedures for the statistical analysis of sample sets. These procedures can be applied in any laboratory environment, using free programs that are platform independent. The scripts are explained and can be adjusted to suit the individual experiment. Finally, a number of example results are given to highlight the use of the statistical analysis in a biological context.

Cross validation of liquid chromatography-mass spectrometry and lectin array for monitoring glycosylation in fed-batch glycoprotein production.

Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC-MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose ß1-, terminal N-acetylglucosamine ß1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose ß1-, while N-acetylglucosamine ß1- was less affected. LC-MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC-MS correlated with the detection of the same epitope by the lectin array.

Negative ion CID fragmentation of O-linked oligosaccharide aldoses--charge induced and charge remote fragmentation.

Collision induced dissociation (CID) fragmentation was compared between reducing and reduced sulfated, sialylated, and neutral O-linked oligosaccharides. It was found that fragmentation of the [M - H](-) ions of aldoses with acidic residues gave unique Z-fragmentation of the reducing end GalNAc containing the acidic C-6 branch, where the entire C-3 branch was lost. This fragmentation pathway, which is not seen in the alditols, showed that the process involved charge remote fragmentation catalyzed by a reducing end acidic anomeric proton. With structures containing sialic acid on both the C-3 and C-6 branch, the [M - H](-) ions were dominated by the loss of sialic acid. This fragmentation pathway was also pronounced in the [M - 2H](2-) ions revealing both the C-6 Z-fragment plus its complementary C-3 C-fragment in addition to glycosidic and cross ring fragmentation. This generation of the Z/C-fragment pairs from GalNAc showed that the charges were not participating in their generation. Fragmentation of neutral aldoses showed pronounced Z-fragmentation believed to be generated by proton migration from the C-6 branch to the negatively charged GalNAc residue followed by charge remote fragmentation similar to the acidic oligosaccharides. In addition, A-type fragments generated by charge induced fragmentation of neutral oligosaccharides were observed when the charge migrated from C-1 of the GalNAc to the GlcNAc residue followed by rearrangement to accommodate the (0,2)A-fragmentation. LC-MS also showed that O-linked aldoses existed as interchangeable α/ß pyranose anomers, in addition to a third isomer (25% of the total free aldose) believed to be the furanose form.