Affinity-capture tandem mass spectrometric characterization of polyprenyl-linked oligosaccharides: tool to study protein N-glycosylation pathways: Anal.Chem

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TypeArticle
Journal titleAnal.Chem
Volume80
Issue14
Pages54685475; # of pages: 8
SubjectACID; analogs & derivatives; analysis; bacterial; biosynthesis; Campylobacter; Campylobacter jejuni; Canada; Capillaries; capillary; chemistry; DIFFERENCE; DISCOVERY; Escherichia; Escherichia coli; ESCHERICHIA-COLI; genetics; Glycomics; Glycosylation; INTERMEDIATE; lipid; Lipids; method; Methods; Mutation; oligosaccharide; Oligosaccharides; PATHWAY; PATHWAYS; protein; PROTEIN GLYCOSYLATION; Proteins; SAMPLES; STRAIN; STRAINS; STRUCTURAL; sugar; SUGARS; SYSTEM; SYSTEMS; Tandem Mass Spectrometry; technique; Tretinoin
AbstractN-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
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberREID2008
NPARC number9360192
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Record identifier47b4f7fd-bc34-429f-92ae-7f6a83f2d285
Record created2009-07-10
Record modified2016-05-09
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