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Generation of Free Oligosaccharides from Bacterial Protein N‐Linked Glycosylation Systems
Author(s) -
Dwivedi Ritika,
Nothaft Harald,
Reiz Bela,
Whittal Randy M.,
Szymanski Christine M.
Publication year - 2013
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.22296
Subject(s) - chemistry , chromatography , derivatization , campylobacter jejuni , periplasmic space , mass spectrometry , biochemistry , bacteria , escherichia coli , gene , biology , genetics
ABSTRACT All Campylobacter species are capable of N ‐glycosylating their proteins and releasing the same oligosaccharides into the periplasm as free oligosaccharides (fOS). Previously, analysis of fOS production in Campylobacter required fOS derivatization or large culture volumes and several chromatography steps prior to fOS analysis. In this study, label‐free fOS extraction and purification methods were developed and coupled with quantitative analysis techniques. Our method follows three simple steps: (1) fOS extraction from the periplasmic space, (2) fOS purification using silica gel chromatography followed by porous graphitized carbon purification and (3) fOS analysis and accurate quantitation using a combination of thin‐layer chromatography, mass spectrometry, NMR, and high performance anion exchange chromatography with pulsed amperometric detection. We applied our techniques to analyze fOS from C . jejuni , C . lari , C. rectus , and C. fetus fetus that produce different fOS structures. We accurately quantified fOS in Campylobacter species that ranged from 7.80 (±0.84) to 49.82 (±0.46) nmoles per gram of wet cell pellet and determined that the C. jejuni fOS comprises 2.5% of the dry cell weight. In addition, a novel di‐phosphorylated fOS species was identified in C. lari . This method provides a sensitive and quantitative method to investigate the genesis, biology and breakdown of fOS in the bacterial N ‐glycosylation systems. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 772–783, 2013.