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-fli ght analyzer has been used to analyze bacterial lipooligosaccharides ( LOS). Crude LOS preparations from pathogenic strains of Haemophilus in fluenzae and Haemophilus ducreyi and a commercial preparation of Lipop olysaccharide 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 cocrystal lization with standard MALDI matrices such as 2,5-dihydroxybenzoic aci d and 1-hydroxyisoquinoline. Under continuous extraction conditions, O -deacylated LOS yielded broad peaks with abundant salt adducts as well as forming prompt fragments through beta-elimination of phosphoric ac id, that is, [M-H3PO4-H]. However, when a time delay was used between ionization and extraction (''delayed extraction'') a significant impro vement was seen in both mass resolution and the stability of the molec ular ions against beta-elimination of phosphoric acid, especially in t he negative-ion mode. Both an external two-point calibration and an in ternal single-point calibration were used to assign masses, the latter of which provided the highest degree of accuracy (better than 0.01% i n most cases). At higher laser powers, the LOS molecules cleave readil y between the oligosaccharide and lipid A moieties yielding a number o f prompt fragments. Postsource decay (PSD) analysis of selected molecu lar ions provided a set of fragments similar to those seen in the line ar spectra, although they were more limited in number because they wer e derived from a single LOS-glycoform. Both the prompt and PSD fragmen ts provided important structural information, especially in assigning the phosphate and phosphoethanolamine substitution pattern of the lipi d A and oligosaccharide portions of LOS. Last, with the addition of et hylenediamineletraacetic acid followed by pulsed sonication, the relat ively insoluble (and impure) LOS preparations yielded MALDI spectra si milar to the O-deacylated LOS, although these intact LOS preparations required higher laser powers to ionize and were generally more affecte d by competing impurities. (C) 1997 American Society for Mass Spectrom etry.