Enhancement of sample loadings for the analysis of oligosaccharides isolated from Pseudomonas aeruginosa using transient isotachophoresis and capillary zone electrophoresis-electrospray-mass spectrometry

The analysis of underivatized core oligosaccharides arising from mild acid hydrolysis of lipopolysaccharides from Pseudomonas aeruginosa serotype O5 was achieved using a transient isotachophoretic preconcentration method coupled to capillary zone electrophoresis-electrospray-mass spectrometry (tCITP-CZE-ESMS). The combination of a tCITP preconcentration step provided a 10- to 50-fold enhancement of sample loading and a corresponding improvement in sensitivity compared to the conventional zone electrophoresis format. Electrophoretic conditions, enabling the separation of these anionic analytes, were developed to determine possible sites of heterogeneity on either the core or the O-chain structures. The tCITP-CZE-ES-MS technique provided unparalleled resolution of the different core glycoforms and oligosaccharides obtained from the acid cleavage of the native endotoxins whether isolated following conventional gel permeation chromatography or obtained from direct hydrolysis of the bacterial isolates. These investigations also highlighted the highly phosphorylated nature of these complex cell membrane components, where the heptose residues of the core oligosaccharide can bear up to six phosphate groups**.