title: Fractionation and analysis of lipopolysaccharide-derived oligosaccharides by zwitterionic-type hydrophilic interaction liquid chromatography coupled with electrospray ionisation mass spectrometry.
abstract: Lipopolysaccharide (LPS, endotoxin) is a main surface antigen and virulence factor of Gram-negative bacteria. Regardless of the source of LPS, this molecule, isolated from the smooth forms of bacteria, is characterised by a general structural layout encompassing three regions: (i) an O-specific polysaccharide (O-PS) - a polymer of repeating oligosaccharide units, (ii) core oligosaccharide (OS), and (iii) the lipid A anchoring LPS in the outer membrane of the cell envelope of Gram-negative bacteria. Structural analysis usually requires degradation of LPS and further efficient separation of various poly- and oligosaccharide glycoforms. The hydrophilic interaction liquid chromatography (HILIC) was shown as an efficient technique for separation of labelled or native neutral and acidic glycans, glycopeptides, sialylated glycans, glycosylated and nonglycosylated peptides. Herein we adopted ZIC(®) (zwitterionic stationary phase covalently attached to porous silica)-HILIC technology in combination with electrospray ionisation mass spectrometry to separate different LPS-derived oligosaccharides. As a result three effective procedures have been developed: (i) to separate different core oligosaccharides of Escherichia coli R1 LOS, (ii) to separate RU-[Hep]-Kdo oligosaccharides from core OS glycoforms of Hafnia alvei PCM 1200 LPS, and (iii) to separate Hep and Kdo-containing mono, di-, tri- and tetrasaccharides of H. alvei PCM 1200 LPS. Moreover, some of developed analytical procedures were scaled to semi-preparative protocols and used to obtain highly-purified fractions of the interest in larger quantities required for future evaluation, analysis, and biological applications.
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Man-Kupisinska
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Bobko
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland.
authors.forename: Tomasz K
authors.initials: TK
authors.lastname: Gozdziewicz
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Maciejewska
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Jachymek
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Lugowski
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, PL-53-114 Wroclaw, Poland. Electronic address: jolanta.lukasiewicz@iitd.pan.wroc.pl.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Lukasiewicz
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis; D005591:Chemical Fractionation; D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D020611:Hafnia alvei / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008070:Lipopolysaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D009844:Oligosaccharides
keywords: Core oligosaccharide; Electrospray ionisation mass spectrometry; Hydrophilic interaction liquid chromatography; Lipopolysaccharide; Zwitterionic stationary phase