Affinity-capture tandem mass spectrometric characterization of polyprenyl-linked oligosaccharides: tool to study protein N-glycosylation pathways.
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 before 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 involve 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 a quick, accurate, 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 the 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.
National Research Council, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6.
Christopher W
CW
Reid
Jacek
J
Stupak
Mark M
MM
Chen
Barbara
B
Imperiali
Jianjun
J
Li
Christine M
CM
Szymanski
D016123:Campylobacter jejuni / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry; D006031:Glycosylation; D008055:Lipids / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000737:chemistry
D008055:Lipids; D009844:Oligosaccharides; D014212:Tretinoin; C034534:3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid