Characterization of bacterial lipooligosaccharides by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) with a time-of-flight analyzer has been used to analyze bacterial lipooligosaccharides (LOS). Crude LOS preparations from pathogenic strains of Haemophilus influenzae and Haemophilus ducreyi and a commercial preparation of lipopolysaccharide from Salmonella typhimurium were treated with hydrazine to remove O-linked fatty acids on the lipid A moiety. The resulting O-deacylated LOS forms were water soluble and more amenable to cocrystallization with standard MALDI matrices such as 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline. Under continuous extraction conditions, O-deacylated LOS yielded broad peaks with abundant salt adducts as well as forming prompt fragments through β-elimination of phosphoric acid, that is, M-H₃PO₄-H]. However, when a time delay was used between ionization and extraction (“delayed extraction”) a significant improvement was seen in both mass resolution and the stability of the molecular ions against β-elimination of phosphoric acid, especially in the negative-ion mode. Both an external two-point calibration and an internal single-point calibration were used to assign masses, the latter of which provided the highest degree of accuracy (better than 0.01% in most cases). At higher laser powers, the LOS molecules cleave readily between the oligosaccharide and lipid A moieties yielding a number of prompt fragments. Postsource decay (PSD) analysis of selected molecular ions provided a set of fragments similar to those seen in the linear spectra, although they were more limited in number because they were derived from a single LOS-glycoform. Both the prompt and PSD fragments provided important structural information, especially in assigning the phosphate and phosphoethanolamine substitution pattern of the lipid A and oligosaccharide portions of LOS. Last, with the addition of ethylenediaminetetraacetic acid followed by pulsed sonication, the relatively insoluble (and impure) LOS preparations yielded MALDI spectra similar to the O-deacylated LOS, although these intact LOS preparations required higher laser powers to ionize and were generally more affected by competing impurities.