Separation and identification of neisserial lipooligosaccharide oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection |
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| Authors: | Swanson Karen V Griffiss J McLeod |
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| Institution: | Centre for Immunochemistry, VA Medical Center (111W1), Department of Laboratory Medicine, University of California San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA. |
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| Abstract: | We determined the optimal conditions for high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) of oligosaccharides (OS) released from neisserial lipooligosaccharides (LOS) by mild acid hydrolysis. We efficiently obtained detailed composition, sequence, and linkage information about high Mr LOS. We found that HPAE-PAD can discriminate isobaric (same Mr) molecules of different structure, for example, nLc4 and Gb4, distinguish alpha from beta chain extensions, and determine the number of phosphoethanolamine (PEA) substituents. HPAE-PAD provided quantitative information that could be used to compare the relative abundances of OS. We used HPAE-PAD to identify all of the known LOS alpha chain antennae. When used with antibody-binding profiles and exoglycosidase digestion results, HPAE-PAD can provide nearly complete structures rapidly. |
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| Keywords: | HPAE-PAD high-performance anion-exchange chromatography with pulsed amperometric detection LOS lipooligosaccharides OS oligosaccharides GSL glycosphingolipids PEA phosphoethanolamine Kdo 2-keto-3-deoxyoctulosonic acid Hep heptose Glc glucose Gal galactose HexNAc N-acetyl hexosamine GlcNAc N-acetyl glucosamine GalNAc N-acetyl galactosamine MALDI-TOF MS matrix-assisted laser desorption and ionization time-of-flight mass spectrometry NMR nuclear magnetic resonance spectroscopy SDS-PAGE Na dodecylsulfate polyacrylamide gel electrophoresis mAb monoclonal antibody |
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