Liquid chromatography ion trap mass spectrometric analysis of oligosaccharides using permethylated derivatives

Reversed phase liquid chromatography was combined with the multiple stage m ass analysis capability of an ion trap mass spectrometer for the characteri zation of permethylated oligosaccharide mixtures. The new method was used t o separate the components of an unlabeled permethylated maltooligomer ladde r, a 2-aminobenzamide-labeled (2-AB) maltooligomer ladder, a complex mixtur e of 2AB-labeled bi- (B), tri- (T), and tetraantennary (Q) standards, and a mixture of recombinant glycoprotein carbohydrates from soluble CD4 with va rying sialic acid (S) content. Using reversed phase HPLC, permethylated mix ture components including alpha and beta anomers were separated based on th eir structures. Fluorescent labeling with 2-aminobenzamide prior to permeth ylation was employed for off-line method development, but was not necessari ly required for mass spectral analysis, as permethylation alone improved th e ionization and fragmentation characteristics of the molecules. Antennae c omposition of permethylated derivatives was determined in MS2 where the fra gmentation patterns of the Y- and B-ion series predominated, and then furth er evaluated in MS3, which provided additional information on branching obt ained from A and X cross-ring fragmentation. Copyright (C) 2001 John Wiley & Sons, Ltd.