Diversity of non-stoichiometric substitutions on the lipopolysaccharide of E. coli C demonstrated by electrospray ionization single quadrupole mass spectrometry.

The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non-stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3-deoxy-D-manno-2-octulosonic acid (KDO) on the R-core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI-Q-MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O-deacylated derivative showed multiple peaks of [M-3H](3-) ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex(5) Hep(3) KDO(2) GlcN(2) C14:0(3-OH)(2). The major components of the O,N-deacylated derivative were the species associated with four and five phosphates on Hex(5) Hep(3) KDO(2) GlcN(2). The polysaccharide portion of LPS also revealed species which corresponded to Hex(5) Hep(3) KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS.