GlycoDigest Guide (UniCarbKB)

alternativeTime edited this page Mar 25, 2014 · 7 revisions


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.

GlycoDigest is 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.

Please read and follow the instructions below otherwise follow link to get started.

Exoglycosidase Preparation and Use Cases

For a detailed description of how to prepare and design exoglycosidases experiments refer to 'Detailed Structural Analysis of N-Glycans Released From Glycoproteins in SDS-PAGE Gel Bands Using HPLC Combined With Exoglycosidase Array Digestions' by Louise Royle, Catherine M. Radcliffe, Raymond A. Dwek, and Pauline M. Rudd (Methods in Molecular Biology)

Liaqat Ali et al., have published guidelines for using exoglycosidases to structurally characterise O-links using LC-MS/MS 'Structural Identification of O-Linked Oligosaccharides Using Exoglycosidases and MSn Together with UniCarb-DB Fragment Spectra Comparison' (Metabolites).

Further information on the action of exoglycosidases with example digestion arrays can be accessed here - a pictorial representation can be viewed

Step One: Build Glycan Structure

We have incorporated GlycanBuilder (Vaadin Release) into UniCarbKB-GlycoDigest. The latest version of GlycanBuilder offers the same functionality previously developed by EUROCarbDB. You can (i) build a new structure, (ii) extend a structure from a predefined list or (iii) build a substructure/epitope. Follow link to get started.

The figure above shows the layout of GlycanBuilder with an N-linked bisecting structure. Structures can be built by adding new monosaccharides or pre-defined groups to the highlighted residue. New groups can be either selected from the medium toolbar or from the structures menu. The list of structural constituents comprises an exhaustive collection of saccharides, substituents, reducing-end markers and saccharide modifications. All the stereo-chemical information about a saccharide, like anomeric conformation, chirality, ring configuration and linkage position, can be specified.

When building glycan structures, to predict the action of exoglycosidase, you must remember to add the anomeric form and linkages between residues. Exoglycosidases are highly-specific and the rules embedded in GlycoDigest will only work if the glycan structure is complete. To specify linkage and anomeric form select a residue and use the 1st Linkage link to configure.

When happy with the structure select the Digest button...

Step Two: Selecting Exoglycosidases

The next step is to theoretical digest the structure with a defined array of exoglycosidases. We have built a library of exoglycosidase rules and reactions that have been experimentally observed. An explanation of the exoglycosidases and example

Careful design and formulation of exoglycosidase arrays is critical to the success of correct glycan structural assignments. Royle L et al., Methods in Molecular Biology Chapter describes how a combination of high-performance liquid chromatography (HPLC) and exoglycosidase enzyme array digestions can deliver quantitative glycan analysis of sugars released from glycoproteins. The chapter provides a complete experimental protocol for preparing and using exoglycosidases.

To create an array of exoglycosidases simply use the 'Build Exoglycosidase Array' box to select the supported exoglycosidase (see below).

The 'short names' for the exoglycosidases are commonly used, however, to help researchers a pop-up table is available that summarises the specificity of the exoglycosidases. To access the table select the 'Information on Exoglycosidases' button.

Once your happy with the enzyme selections press 'Digest'.

Step Three: Results

GlycoDigest will now simulate the combinations of exoglycosidase on the submitted structure, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. When complete the 'Results' section will be updated with the end-product(s). Each product-glycan is shown accompained by the relevant combination of exoglycosidase(s).

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