O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery

Abstract

O-glycosylation is a post-translational modification of proteins crucial to molecular mechanisms in health and disease. O-glycans are typically highly heterogeneous. The involvement of specific O-glycan sequences in many bio-recognition systems is yet to be determined because of a lack of efficient methodologies. We describe here a targeted microarray approach: O-glycome beam search that is both robust and efficient for O-glycan ligand-discovery. Substantial simplification of the complex O-glycome profile and facile chromatographic resolution is achieved by arraying O-glycans as branches, monitoring by mass spectrometry, focusing on promising fractions, and on-array immuno-sequencing. This is orders of magnitude more sensitive than traditional methods. We have applied beam search approach to porcine stomach mucin and identified extremely minor components previously undetected within the O-glycome of this mucin that are ligands for the adhesive proteins of two rotaviruses. The approach is applicable to O-glycome recognition studies in a wide range of biological settings to give insights into glycan recognition structures in natural microenvironments. Molecular & Cellular

The neoglycolipid (NGL) technology‐based microarrays and future prospects

“…The latest advance of the NGL technology has been the Beam Search approach developed as a highly targeted process to discover natural O-glycan ligands [78]. This approach takes advantage of robotic arraying of natural O-glycome NGL fractions for analyses of their ligand expression, to focus on promising fractions.…”

“…There are similarities in the aims of the Beam Search approach [78] and that of the 'Shotgun' glycomics of Cummings and colleagues [80,81]. Both are to characterize ligands among glycans from natural sources, using sequence analyses by mass spectrometry and applying sequence-specific lectins and antibodies.…”

“…There are differences, however, in the design and application of the two approaches. As we have discussed [78], the shotgun glycomics involves the dissection of the glycome by multidimensional HPLC fractionation aiming to separate individual glycans 'as homogeneous as possible' in the first place for printing. Particular biological questions and applications often come at a later stage.…”

“…detected by MALDI MS in middle fraction 17: F1H3N2-OX ([M-H] -2093) & F1H2N2-OY ([M-H] Exploratory Beam Search O-glycan array of NGLs derived from~1mg neutral O-glycan alditols for initial screening for the ligands of the VP8* protein of rotavirus P[19]. This figure is adapted from reference[78]. In (B) and (C), the Middle and Lower migrating fractions in (A) were subfractionated by HPLC and printed as a primary NGL array with MS monitoring.…”

“…Schematic presentation of the generation of NGLs from an O-glycan alditol. This figure is adapted from reference[78].…”