Structural Elucidation of Intact Rough-type Lipopolysaccharides Using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots

Lipopolysaccharides (LPSs) are a hallmark virulence factor
of Gram-negative
bacteria. They are complex, structurally heterogeneous mixtures due
to variations in number, type, and position of their simplest units:
fatty acids and monosaccharides. Thus, LPS structural characterization
by traditional mass spectrometry (MS) methods is challenging. Here,
we describe the benefits of field asymmetric ion mobility spectrometry
(FAIMS) for analysis of an intact R-type lipopolysaccharide complex
mixture (lipooligosaccharide; LOS). Structural characterization was
performed using Escherichia coli J5
(Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine
research. FAIMS gas-phase fractionation improved the (S/N) ratio and
number of detected LOS species. Additionally, FAIMS allowed the separation
of overlapping isobars facilitating their tandem MS characterization
and unequivocal structural assignments. In addition to FAIMS gas-phase
fractionation benefits, extra sorting of the structurally related
LOS molecules was further accomplished using Kendrick mass defect
(KMD) plots. Notably, a custom KMD base unit of [Na–H] created
a highly organized KMD plot that allowed identification of interesting
and novel structural differences across the different LOS ion families,
i.e., ions with different acylation degrees, oligosaccharides composition,
and chemical modifications. Defining the composition of a single LOS
ion by tandem MS along with the organized KMD plot structural network
was sufficient to deduce the composition of 181 LOS species out of
321 species present in the mixture. The combination of FAIMS and KMD
plots allowed in-depth characterization of the complex LOS mixture
and uncovered a wealth of novel information about its structural variations.