Tagged Glycan Library and Shotgun Glycan Microarray

Chemical Release of glycans from glycoconjugates 

The release of free glycans from glycoconjugates is used for many glycomic analyses.  This process has classically been carried out using harsh chemical processes such as hydrazinolysis to release N-glycans or strong base degradation to release O-glycans from glycoproteins.  Glycolipid oligosaccharides can be released using ozonolysis followed by strong base.  The harsh chemical conditions sometimes result in further degradation of the released glycans. We have developed a proprietary process for chemically releasing glycans from glycoconjugates using controlled oxidation (ORNG). This process rapidly releases glycans and glycan derivatives from all glycoconjugates simultaneously, and the non-degraded glycans can be isolated from the supernatant of the chemically treated homogenate.  Since the mechanism of release is different for each glycosidic linkage, the source of the resulting glycans can be identified based on the nature of the reducing end.  This process is being developed for microscale analysis of glycoconjugates; however, this process is also useful for obtaining large quantities of N- and O-linked glycans and glycolipid-derived glycans from tissues, organs, or other biological samples, because we can use milligram to kilogram quantities of starting material. 

Production of Tagged Glycan Libraries (TGL) 

Released glycans have a functional group at their reducing ends in the form of an aldehyde, a nitrile, or a carboxyl group.  We have developed many methods to attach a variety of bi-functional fluorescent tags to the reducing ends of glycans.  The tagged glycans with fluorescence and a primary amino group can be purified by multi-dimensional HPLC to tagged glycan libraries (TGLs). The TGLs can be stored for later retrieval, immobilized onto solid surfaces such as glycan microarray for functional, and subsequently structurally analyzed by mass spectrometry (MS). 

Glycan Immobilization and Molecular Interaction Analysis 

Glycomic approaches to understanding the relationship between glycan structure and function have classically focused on determining the structures of N-, O- and glycolipid-linked glycans.  However, listing and cataloging structures, like collecting stamps, has really provided little information regarding the function of glycans.  Since the functions of glycans are thought in large part to involve protein-glycan interactions, we have utilized our unique chemistries to address functional Glycomics by investigation protein-glycan interactions.  Immobilizing glycans to solid phases is the first step in this process, and subsequent analyses involving the binding of protein, cells, microorganisms or other species that bind glycans are relatively simple and include detection of labeled organisms or glycan binding molecules, fluorescence-activated cell sorting (FACS) analysis of glycan coated beads, and pull down experiments to identify unknown glycan binging proteins that can be identified by proteomic analyses. 

Development of Shotgun Glycan TGL and Microarray  

We have had over 10 years of experience printing glycans as microarrays on microscope slides that can subsequently be interrogated with fluorescently labeled glycan binding molecules and microorganisms. These glycans can be structurally defined glycans where the data support interpretation of the probably glycan binding specificity of the binding proteins.  On the other hand, the glycans can be arrays of tagged glycan libraries (TGL) that we call Shotgun Glycan Microarrays (SGM) where any glycome may be interrogated using your suspected glycan binding protein or microorganism to identify physiologically relevant glycans that can retrieved from the TGL for more detailed structural analyses.