A new rapid and micro-scale hydrolysis, using triethylamine citrate, for lipopolysaccharide characterization by mass spectrometry.

Endotoxin (lipopolysaccharide, LPS) is, in general, composed of two moieties: a hydrophilic polysaccharide linked to a hydrophobic lipid A terminal unit and forms a major surface component of gram-negative bacteria. The structural features of LPS moieties play a role in pathogenesis and also involve immunogenicity and diagnostic serology. The major toxic factor of LPS resides in the lipid A moiety, anchored in the outer layer of the bacterium, and its relative biological activity is critically related to fine structural features within the molecule. In establishing relationships between structural features and biological activities of LPS it is of the utmost importance to develop new analytical methods that can be applied to the complete unambiguous characterization of a specific LPS molecule. Herein is presented a practical rapid and sensitive analytical procedure for the mass spectral screening of LPS using triethylamine citrate as an agent for both disaggregation and mild hydrolysis of LPS. It provides improved matrix-assisted laser desorption/ionization (MALDI) mass spectra and, in particular, affords the identification of fragments retaining labile substituents present in the native macromolecular LPS structures. The methods were developed and applied using purified LPS of Escherichia coli and Salmonella enterica, as well as more complex LPS of Actnobacillus pleuropneumoniae.