Affinity-capture tandem mass spectrometric characterization of polyprenyl-linked oligosaccharides: a novel tool to study protein N-glycosylation pathways

摘要

N-Glycosylation of proteins is recognized as one of the most common post translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes until the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. To date most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involves the use of radiolabels and chromatographic separation. Technologies capable of characterizing eukaryotic and the newly described bacterial N-glycosylation systems from biologically relevant samples in an accurate, rapid, and cost effective manner are needed. In this paper a new glycomics strategy based on lectin-affinity capture was devised and validated on the C. jejuni N-glycan pathway and with engineered Escherichia coli strains expressing the functional C. jejuni pathway. The lipid-linked oligosaccharide intermediates of the Pgl pathway were then enriched using SBA-agarose affinity-capture and examined by capillary electrophoresis-mass spectrometry (CE-MS). We demonstrate that this method is capable of detecting low levels of LLOs, the sugars are indeed assembled on undecaprenylpyrophosphate, and structural information for expected and unexpected LLOs can be obtained without further sample manipulation. Furthermore, CE-MS analyses of C. jejuni and the E. coli “glyco-factories” showed striking differences in the assembly and control of N-glycan biosynthesis.