Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry.

Lipopolysaccharide (LPS) extracted from the deep rough mutant of Escherichia coli D31m4 was disaggregated with 0.1 M EDTA, pH 7.0, and fractionated on a diethylaminoethyl-cellulose column to yield the biphosphate form of LPS. After methylation, the derivative was purified by reverse-phase high performance liquid chromatography using a C18-bonded silica cartridge. A linear gradient of 50-100% isopropyl alcohol/water (93:7, v/v) in acetonitrile/water (93:7, v/v) was used over a period of 60 min. The derivatized LPS showed a single major peak by high performance liquid chromatography, and this hexamethyl hexaacyl LPS was recovered and subjected to chemical analysis, plasma desorption mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. Chemical analysis of the purified hexamethyl LPS quantitated certain key chemical compositions. Plasma desorption mass spectrometry showed a molecular ion (M + CH2 + Na)+ at m/z 2360, which established the molecular formula and Mr to be C116H214N2O39P2 and 2323, respectively. Thus, it contained two each of glucosamine, 2-keto-3-deoxyoctonate, and phosphate; four beta-hydroxymyristates; one laurate; and one myristate. NMR spectroscopy confirmed the locations of the four ester-linked fatty acyl groups. Based on these results and the known structure of free lipid A, the complete structure of the deep-rough chemotype LPS from E. coli can now be presented with confidence. This is the first report of a successful purification to homogeneity and the characterization of the simplest of the LPS at the intact level. This study shows that the natural distribution of the lipid A moiety of LPS from E. coli D31m4 is hexaacyl/pentaacyl in a molar ratio of greater than 90:less than 10. Acid hydrolysis of LPS causes the formation of the lower homologues of the free lipid A.