Fractionation and analysis of lipopolysaccharide-derived oligosaccharides by zwitterionic-type hydrophilic interaction liquid chromatography coupled with electrospray ionisation mass spectrometry.

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.

Reprint of "new complete structure of Hafnia alvei clinical isolate strain PCM 2670 semi-rough lipopolysaccharide".

Hafnia alvei strain PCM 2670 is a clinical isolate from a patient with chronic reproductive tract infection. The novel structure of the semi-rough lipopolysaccharide was established with the use of NMR spectroscopy and mass spectrometry as well as immunochemical techniques. According to the mass spectrometry data, heptose in the oligosaccharide is partially substituted by glycine. H. alvei PCM 2670 core structure encompasses the common core of H. alvei which is modified with two additional galactose units. [formula see text] The 6-substituted galactose is the O-antigen repeating unit substitution residue. The repeating unit consists of five monosaccharide residues and has the following structure: →2)-ß-Galp-(1→6)-α-Glcp-(1→6)-αGlcpNAc3OAc-(1→4)-α-GalpA-(1→3)-ß-GlcpNAc6OAc-(1→6)-core.

New complete structure of Hafnia alvei clinical isolate strain PCM 2670 semi-rough lipopolysaccharide.

Hafnia alvei strain PCM 2670 is a clinical isolate from a patient with chronic reproductive tract infection. The novel structure of the semi-rough lipopolysaccharide was established with the use of NMR spectroscopy and mass spectrometry as well as immunochemical techniques. According to the mass spectrometry data, heptose in the oligosaccharide is partially substituted by glycine. H. alvei PCM 2670 core structure encompasses the common core of H. alvei which is modified with two additional galactose units. [structure: see text]. The 6-substituted galactose is the O-antigen repeating unit substitution residue. The repeating unit consists of five monosaccharide residues and has the following structure: →2)-ß-Galp-(1→6)-α-Glcp-(1→6)-αGlcpNAc3OAc-(1→4)-α-GalpA-(1→3)-ß-GlcpNAc6OAc-(1→6)-core.