Toward a Platform for Comprehensive Glycan Sequencing

Abstract

From a series of recently published reports, an analytical platform has been proposed for a quantitative and qualitative measure of N-and O-glycosylation, complete with peptide-glycan connectivity and detailed structural understanding. As distant as this may appear, a best methods approach will appear that must move us beyond the cartoon stage of structural understanding. Thus, with this unifying goal in mind, we summarize a series of individually promising first phase protocols of sample preparation (release, purification, and quantification) that remain congruent with a concluding phase (methylation and MS n ) for documented structural detail. Sequential enzymatic Nglycan and chemical O-glycan release from glycopeptides with intervening solid phase extraction and derivatization will provide for a comparative quantification measure of glycosylation. The O-glycan release will be nonreductive and coupled with Michael addition to a pyrazolone analog (1-phenyl-3-methyl-5-pyrazolone) with both the peptide and glycan labeled. The product glycans are stable to methylation and appropriate for sequential disassembly (MS n ). An application using human serum and cancer samples has been detailed characterizing sLe x and comparable valence epitopes. This integrated platform will provide opportunities at variable points to contrast, share, and advance alternative protocols in a collaborative effort that is greatly needed. This integrated platform provides end point opportunities to confirm structural details compiled from synthetic standards and well characterized biologics by MS n . Molecular & Cellular Proteomics 12: 10.1074/mcp.R112.026823, 866 -873, 2013. A UNIFIED GLYCOMICS PLATFORMThe NHLBI, National Institutes of Health, established a Program of Excellence in Glycosciences that supports a study of glycans ubiquitously found on the surfaces of all mammalian cells. Such structures influence a wide variety of cellular and disease-related processes that are governed by the composition and configuration of the interacting partners and the details of structure and conformation within that supramolecular environment. An example of these cohorts are the selectins (E-, L-, and P-) that bind to sialofucosylated ligands displayed on their respective glycans (1, 2). Following an earlier focus by the NIGMS and the NCI of the National Institutes of Health, the NHLBI introduced this Program of Excellence in Glycosciences to bring a more comprehensive glycomics endeavor to support the growing importance of glycan structures in heart, lung, and blood disease research. The Program of Excellence has established three fundamental goals as follows: (i) to develop and expand core facilities across the country; (ii) to facilitate collaborations and distribute research findings, and (iii) to train future generations of scientists to be cognizant in both glycan biology and chemistry. These are laudable goals by any measure, but what blurs the effort and forward momentum must be the considerations of the last goal. With the expanding list of ...

Breast Milk Oligosaccharides: Structure-Function Relationships in the Neonate

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