Analysis of bacterial lipid-linked oligosaccharide intermediates using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry : Heterogeneity in the polyisoprenyl carrier revealed

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DOIResolve DOI: http://doi.org/10.1021/ac9013622
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TypeArticle
Journal titleAnalytical Chemistry
Volume81
Issue20
Pages84728478; # of pages: 7
AbstractN-Glycosylation of proteins is recognized as one of the most common post-translational modifications. It was believed that N-glycosylation occurred exclusively in eukaryotes until the recent discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni, which has similarities to the eukaryotic system and adds proteins en bloc from a lipid carrier to a protein acceptor. In addition to N-linked glycans, a number of pathogenic bacteria such as Pseudomonas aeruginosa and Neisseria species have been shown to O-glycosylate their proteins through polyisoprene-linked intermediates. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. With the increasing frequency of reports of bacterial protein glycosylation that proceed through lipid-mediated steps, there is a need for technologies capable of characterizing these newly described bacterial systems as well as eukaryotic pathways from biologically relevant samples in an accurate, rapid, and cost-effective manner. In this paper, a new glycomics strategy based on porous graphite carbon (PGC) liquid chromatography mass spectrometry (LC-MS) was devised and validated on the C. jejuni N-glycan pathway. Lipidlinked oligosaccharide intermediates of the Pgl pathway from crude lipid extracts were separated using online chromatography on a capillary PGC column with a chloroform gradient. By exploiting the retention properties of hydrophobic and polar analytes on PGC, baseline separation of LLOs with minor changes in oligosaccharide structure and polyisoprene chain length was obtained. This method is capable of analyzing low levels of LLOs (from approximately 10⁶ bacterial cells) and distinguishing the LLOs that differ by as little as one monosaccharide or polyisoprene unit. Furthermore, we have demonstrated for the first time that oligosaccharides of the C. jejuni Pgl pathway are assembled on different polyisoprenes, e. g. C₄₅, C₆₀, and apparent hydroxylated forms, in addition to those previously reported (i.e., C₅₀ and C₅₅). The hydroxylated forms of the LLOs are believed to be an intermediate in the degradation of accumulated LLOs for polyisoprene carrier recycling.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Institute for Biological Sciences
Peer reviewedYes
NPARC number15551524
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Record identifier25c41d16-016d-4ba0-850b-4dde66de4f43
Record created2010-08-16
Record modified2016-05-09
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