INVESTIGATION OF THE STRUCTURAL HETEROGENEITY OF LIPOOLIGOSACCHARIDESFROM PATHOGENIC HAEMOPHILUS AND NEISSERIA SPECIES AND OF R-TYPE LIPOPOLYSACCHARIDES FROM SALMONELLA-TYPHIMURIUM BY ELECTROSPRAY MASS-SPECTROMETRY

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophi lus and Neisseria species is evident from the multiplicity of componen ts observed with electrophoretic analyses. Knowledge of the precise st ructures that make up these diverse LOS molecules is clearly the key t o reaching an understanding of pathogenic processes such as phase vari ation and molecular mimicry. Except for a few cases, little is known a bout the specific structural features of LOS that underlie phase varia tion and molecular mimicry, partly because of the inherent difficultie s in the structural elucidation of these complex glycolipids. In the l ipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia c oli, rough, or R-type, mutants have been isolated that have provided i nsight into the biosynthetic pathways and associated genetics that con trol LPS expression. Nonetheless, recent work has shown that these R-t ype LPS are more complex than originally thought, and significant hete rogeneity is still observed, primarily in their phosphorylation states . In order to investigate the structures of LPS and LOS in a more rapi d fashion, we have determined the precise molecular weights of LOS (an d LPS) preparations from various Haemophilus, Neisseria, and Salmonell a species by electrospray ionization-mass spectrometry. The LOS (or LP S) were first O-deacylated under mild hydrazine conditions to remove O -linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M -nH)n-, where n refers to the number of protons rem oved and therefore determines the absolute charge state, n = z. Mass s pectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heteroge neity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the v ariable phosphorylation of the core of S. typhimurium LPS have afforde d insights into the biosynthetic pathways used by these organisms. Inf ormation of this type is important for understanding the underlying ge netic and environmental factors controlling LOS and LPS expression.