Summary: Sequencing oligosaccharides by exoglycosidases, either sequentially or in an array format, is a powerful tool to unambiguously determine the structure of complex N- and O-link glycans. Here, we introduce GlycoDigest, a tool that simulates exoglycosidase digestion, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. The tool allows the targeted design of glycosidase enzyme mixtures by allowing researchers to model the action of exoglycosidases, thereby validating and improving the efficiency and accuracy of glycan analysis. Availability and implementation: http://www.glycodigest.org. Contact: matthew.campbell@mq.edu.au or frederique.lisacek@isb-sib.c

Abrahams, Jodie L.

Campbell, Matthew P.

Gotz, Lou

Karlsson, Niclas G.

Lisacek, Frederique

Mariethoz, Julien

Packer, Nicolle H.

Rudd, Pauline M.

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Vol. 30 no. 21 2014, pages 3131–3133BIOINFORMATICS APPLICATIONS NOTE doi:10.1093/bioinformatics/btu425Structural bioinformatics Advance Access publication July 10, 2014GlycoDigest: a tool for the targeted use of exoglycosidasedigestions in glycan structure determinationLou Gotz1, Jodie L. Abrahams2, Julien Mariethoz1, Pauline M. Rudd3, Niclas G. Karlsson4,Nicolle H. Packer2, Matthew P. Campbell2,* and Frederique Lisacek1,5,*1Proteome Informatics Group, Swiss Institute of Bioinformatics, 1211 Geneva, Switzerland, 2Biomolecular FrontiersResearch Centre, Macquarie University, North Ryde, NSW 2109, Australia, 3National Institute for Bioprocessing Researchand Training, GlycoScience Group, Dublin, Ireland, 4Department of Medical Biochemistry and Cell Biology, University ofGothenburg, 40530 Gothenburg, Sweden and 5Section of Biology, University of Geneva, 1211 Geneva, SwitzerlandAssociate Editor: Janet KelsoABSTRACTSummary: Sequencing oligosaccharides by exoglycosidases, eithersequentially or in an array format, is a powerful tool to unambiguouslydetermine the structure of complex N- and O-link glycans. Here, weintroduce GlycoDigest, a tool that simulates exoglycosidase digestion,based on controlled rules acquired from expert knowledge and ex-perimental evidence available in GlycoBase. The tool allows the tar-geted design of glycosidase enzyme mixtures by allowing researchersto model the action of exoglycosidases, thereby validating and im-proving the efficiency and accuracy of glycan analysis.Availability and implementation: http://www.glycodigest.org.Contact: matthew.campbell@mq.edu.au orfrederique.lisacek@isb-sib.chReceived on February 17, 2014; revised on April 29, 2014; acceptedon June 25, 20141 INTRODUCTIONProtein glycosylation plays a key role in a wide range of biolo-gical processes. Advances in glycansequencing technology showthat glycoproteins and glycolipids exist in many glycosylatedvariants, or glycoforms, which can differ substantially in theirbiochemical properties and functions. Glycans are structurallydiverse, incorporating a wide range of monosaccharide residuesand glycosidic linkages. Given that protein glycosylation isinvolved in numerous cellular processes and is implicated in dis-ease progression, the ability to accurately characterize glycanstructures and substructures and the determination of the func-tion of these modifications, is increasingly important. However,the difficulty associated with oligosaccharide sequencing has re-sulted in researchers using only their knowledge of the biosyn-thetic pathways for protein glycosylation to report proposedglycan structures.Various analytical platforms are available for the sensitive andefficient analysis of N- and O-glycans. Such strategies includechromatographic and electrophoretic separations (with fluores-cence detection), mass spectrometry (MS) and a combination ofboth. However, each particular methodology has its strengthsand weaknesses, especially in terms of the information content(structural features) that can be readily measured. For example,limitations in material, the lack of fragments and resolution/separation of peaks may yield only partial structures wherebythe linkage, the identity or sequence of residue(s) cannot befully identified. The use of orthogonal methodologies for struc-tural and functional glycomics overcomes these constraints butoften necessitates the integration of results from multiple com-plementary tools.1.1 Exoglycosidase digestions of oligosaccharidesOne of the most commonly used methods for determining thesequence and structure of oligosaccharides is the enzymatic ana-lysis of oligosaccharides using highly specific exoglycosidases,either sequentially or in array form. Enzymatic methods havethe ability to determine, in many cases unambiguously, fulland accurate sequence and linkage information. As describedby (Kobata, 1979), they are highly specific to anomeric config-uration and often to monosaccharide type and linkage. Some arealso specific to other structural features, such as local and distantsaccharide branching. This approach has been extensively re-ported for analysis of fluorescently labeled N-linked oligosac-charides (and to a lesser degree for O-links) in which treatmentwith known enzymatic specificities, followed by chromato-graphic separation and observation of shifts in retention time,is used to validate individual glycan structural features (Guileet al., 1996). Furthermore, exoglycosidases combined with MShave been demonstrated to be effective for yielding structuralinformation (Ali et al., 2012).GlycoDigest is a tool that simulates the action of these en-zymes on released oligosaccharides. It has been developed toassist glycobiologists design mixtures of exoglycosidases thatcan be used to guide the precise determination of glycanstructures.2 DESIGN AND IMPLEMENTATIONGlycoDigest is available in three formats: (i) stand-alone Javaapplication, (ii) web-based Java applet (http://glycodigest.org/digest.html) and (iii) an integrated feature of UniCarbKB(http://unicarbkb.org/glycodigest) and GlycoBase databases;links are provided for each fully defined structure. The applica-tion is written in Java 7.0, and the source code is available at*To whom correspondence should be addressed. The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 3131https://bitbucket.org/sib-pig. The stand-alone version is a Swing-based application built with the MigLayout layout manager,which can be run on all operating systems. GlycoDigest is avail-able as a Java library that is integrated with UniCarbKB, allow-ing users to theoretically digestN- andO-linked glycan structuresstored in this curated database. GlycanBuilder (Damerell et al.,2012) acts as the interface for all GlycoDigest variants, allowingusers to create structures by sequentially adding monosacchar-ides, modifications or reducing-end markers. The application in-tegrates libraries developed by EUROCarbDB and GlycomeDBincluding universal formats for encoding of glycan structures(Campbell et al., 2014a).3 EXOGLYCOSIDASE KNOWLEDGEBASECareful preparation and formulation of exoglycosidase arrays iscritical to accurate glycan structural assignments, and strategiesfor designing digestions are detailed (Royle et al., 2006). Becauseof the structural complexity of glycans, i.e. degree of branchingand steric hindrance, the sequencing of particular motifs is oftendependent upon the correct combination of exoglycosidases. Forexample, the Fuc(1-3) residue of the Lewis x epitope can onlybe cleaved after treatment with -galactosidase. Knowledgeof exoglycosidase reactions has been acquired by extensiveanalytical experience and the creation of suitable experimentaldatabases. GlycoBase is an High Performance LiquidChromatography (HPLC) retention time database of experimen-tally characterized glycan structures, which provides comprehen-sive information on known exoglycosidase digestions (Campbellet al., 2008). This wealth of information builds upon the methodsused by the reagent array analysis method (Prime and Merry,1998). From this base, the rules included in GlycoDigest areexperimental observations on the behavior and mode of actionof exoglycosidases.4 USING GLYCODIGEST4.1 GlycoDigest desktop applicationThe stand-alone release offers a user-friendly interface consistingof three components: (i) glycan sequences can be directly enteredinto the ‘load’ panel in genuine or Consortium for FunctionalGlycomics (CFG) modified, International Union of Pure andApplied Chemistry (IUPAC) condensed or GlycoCT format,or built using the embedded version of GlycanBuilder; (ii) acombination of exoglycosidase enzymes can be selected fromthe middle panel, and the input structure is processed in silicousing the rules explained above; (iii) the theoretical results of theenzymatic digestion are returned in GlycoCT format and dis-played in the ‘Simulation Results’ panel (Fig. 1). Graphical rep-resentations of the glycan structures can be displayed using theimage rendering Application Programming Interface (API),which are linked to UniCarbKB entries.4.2 GlycoDigest web versionA Java applet version of GlycoDigest uses the same functionalityand interface described above. However, the structure encodingentries in Panels 1 and 3 are displayed in the same page asopposed to being displayed in new windows. As the stand-aloneversion, known structures are linked to UniCarbKB entries.4.3 Integrating GlycoDigest with UniCarbKBUniCarbKB offers public access to a growing, curated databaseof information on the glycan structures on glycoproteins. Foreach N- and O-link glycan structure with defined linkage infor-mation, a ‘GlycoDigest’ link provides users the option to select acombination of enzymes for simulating exoglycosidase treat-ment(s). Proposed structures for each undigested and digestedglycan resulting from the action of the exoglycosidases can besearched in UniCarbKB (Campbell et al., 2014b) for associatedpublished and experimental information, and for combined exo-glycosidase-MS/MS characterization of oligosaccharides (Aliet al., 2012) using UniCarb-DB spectra (Hayes et al., 2011).Alternatively, digestions can be run on structures built usingGlycanBuilder.5 CONCLUSIONSGlycoDigest fills the need for a robust and intuitive means forglycobiologists to simulate and design mixtures of exoglycosi-dases with which to experimentally validate proposed glycanstructures. The tool minimizes any chance of user bias in inter-preting what may be ambiguous results and ensures that allstructures compatible with the experimental data are considered.The availability of the GlycoDigest Java library will allow bioin-formaticians the option of integrating this tool for managing theinput and output of exoglycosidase-treated glycan structures.Funding: Swiss National Science Foundation (31003A_141215);Macquarie University Research Excellence Scheme PostgraduateScholarship; The Australian National eResearch CollaborationTools and Resources project (RT016).Conflict of interest: none declared.REFERENCESAli,L. et al. (2012) Structural identification of O-linked oligosaccharides using exo-glycosidases and MSn together with UniCarb-DB fragment spectra comparison.Metabolites, 2, 648–666.Campbell,M.P. et al. (2008) GlycoBase and autoGU: tools for HPLC-based glycananalysis. Bioinformatics 2008, 24, 1214–1216.Fig. 1. Interface for the desktop version of GlycoDigest with examples ofinput/output glycan structures displayed in the essentials ofGlycobiology/CFG format3132L.Gotz et al.Campbell,M.P. et al. (2014a) Toolboxes for a standardised and systematic study ofglycans. BMC Bioinformatics, 15 (Suppl 1), S9.Campbell,M.P. et al. (2014b) UniCarbKB: building a knowledge platform for gly-coproteomics. Nucleic Acids Res, 42, D215–D221.Damerell,D. et al. (2012) The GlycanBuilder and GlycoWorkbench glycoinfor-matics tools: updates and new developments. Biol. Chem., 393, 1357–1362.Guile,G.R. et al. (1996) A rapid high-resolution high-performance liquid chroma-tographic method for separating glycan mixtures and analyzing oligosaccharideprofiles. Anal Biochem, 240, 210–226.Hayes,C.A. et al. (2011) UniCarb-DB: a database resource for glycomic discovery.Bioinformatics, 27, 1343–1344.Kobata,A. (1979) Use of endo- and exoglycosidases for structural studies of glyco-conjugates. Anal. Biochem., 100, 1–14.Prime,S. and Merry,T. (1998) Exoglycosidase sequencing of N-linked glycans by thereagent array analysis method (RAAM). Methods Mol. Biol., 76, 53–69.Royle,L. et al. (2006) Detailed structural analysis of N-glycans released from glyco-proteins in SDS-PAGE gel bands using HPLC combined with exoglycosidasearray digestions. Methods Mol. Biol., 347, 125–143.3133GlycoDigest

GlycoDigest: a tool for the targeted use of exoglycosidase digestions in glycan structure determination

GlycoDigest: a tool for the targeted use of exoglycosidase digestions in glycan structure determination

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