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 ser otype O5 was achieved using a transient isotachophoretic preconcentrat ion method coupled to capillary zone electrophoresis-electrospray-mass spectrometry (tCITP-CZE-ESMS). The combination of a tCITP preconcentr ation step provided a 10- to 50-fold enhancement of sample loading and a corresponding improvement in sensitivity compared to the convention al zone electrophoresis format. Electrophoretic conditions, enabling t he separation of these anionic analytes, were developed to determine p ossible sites of heterogeneity on either the core or the O-chain struc tures. The tCITP-CZE-ES-MS technique provided unparalleled resolution of the different core glycoforms and oligosaccharides obtained from th e acid cleavage of the native endotoxins whether isolated following co nventional gel permeation chromatography or obtained from direct hydro lysis of the bacterial isolates. These investigations also highlighted the highly phosphorylated nature of these complex cell membrane compo nents, where the heptose residues of the core oligosaccharide can bear up to six phosphate groups*.