Abstract
The isolation, purification and analysis of the lipid A obtained from Mesorhizobium loti Ayac 1 BII strain is presented. Analysis of the carbohydrate moiety after acid hydrolysis by high-pH anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD) showed the presence of glucosamine and galacturonic acid as the only sugar components. Gas chromatographic (GC) and GC/mass spectrometric (MS) analysis of the fatty acids revealed the presence of 3-OH-C12:0; 3-OH-C13:0; 3-OH-C20:0 and 27-OH-C28:0 among the major hydroxylated species. In addition, C16:0, C17:0, C18:0 and C 20:0 were shown as main saturated fatty acids. Different polyacylated species were evidenced by thin layer chromatography of lipid A, allowing the purification of two fractions. Ultraviolet matrix-assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) MS analysis with different matrices, in the positive- and negative-ion mode, was performed. The fast moving component revealed the presence of hexa-acylated species, varying in the fatty acid composition. Species containing three 3-OH fatty acids and a 27-OH-C28:0 fatty acid were observed. Individual ions within this family differ by +/-14 mass units. The slow moving component was enriched mainly in penta-acylated species. Among them, three subgroups were detected: the major one compatible with the sugar core bearing two 3-OH 20:0 fatty acids, a 3-OH 13:0 or a 3-OH 12:0 fatty acid, a 27-OH 28:0 fatty acid and one saturated fatty acid. Each signal differs in a C18:0 acyl unit from the corresponding hexa-acylated family. On the other hand, a subgroup bearing one 3-OH 20:0 fatty acid, one 27-OH 28:0 fatty acid and two non-polar fatty acids was shown. A minor subgroup compatible with structures containing two hydroxylated and three non-polar fatty acids was also detected. The results obtained showed that nor-harmane was an excellent matrix for charged lipid A structural studies in both, positive and negative ion modes.
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