Substitution pattern of 3-deoxy-D-manno-oct-2-ulosonic acid in bacterial lipopolysaccharides investigated by methylation analysis of whole LPS.
3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a constituent of the inner core part of bacterial lipopolysaccharides (LPS). This sugar may contribute to biological activities of the LPS, the type of substitution of Kdo is thus of importance and this work is aimed at the evaluation of a method for monitoring the substitution of Kdo in LPS. The procedure consists of three steps, namely permethylation of the lipopolysaccharide, with iodomethane and sodium methylsulfinylmethanide or NaOH in Me(2)SO, or with methyl triflate, then the product is methanolysed with HCl in MeOH and acetylated with acetic anhydride in pyridine. The resulting partially methylated acetates of Kdo methyl glycosides were analyzed by gas-liquid chromatography-electron impact ionization mass spectrometry (GLC-MS). For several derivatives of Kdo, specific GLC retention times and MS fragmentation patterns were determined. Lipopolysaccharides from several bacterial strains were isolated and analyzed with three different methods of methylation. The complete solubilization of the LPS in the acid form allows diminishing possible undermethylation. Sodium methylsulfinylmethanide is the most efficient agent in the permethylation of the whole LPS, of all the tested procedures. Methylation with methyl triflate allows the detection of base labile substituents on Kdo residues.
Rybka
Jacek
J
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, PL-53 114, Wrocław, Poland.
Zielińska-Kuźniarz
Katarzyna
K
Korzeniowska-Kowal
Agnieszka
A
Sondej
Aneta
A
Gamian
Andrzej
A
D002849:Chromatography, Gas; D004731:Endotoxins / Q000737:chemistry; D006847:Hydrocarbons, Iodinated / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry*; D013058:Mass Spectrometry; D008697:Methane / Q000737:chemistry; D008745:Methylation; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D012972:Sodium Hydroxide / Q000737:chemistry; D013400:Sugar Acids / Q000737:chemistry*
D004731:Endotoxins; D006847:Hydrocarbons, Iodinated; D008070:Lipopolysaccharides; D011135:Polysaccharides, Bacterial; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; D012972:Sodium Hydroxide; C014055:methyl iodide; D008697:Methane