ISOMERIC DIFFERENTIATION OF ASPARAGINE-LINKED OLIGOSACCHARIDES BY MATRIX-ASSISTED LASER DESORPTION-IONIZATION POSTSOURCE DECAY TIME-OF-FLIGHT MASS-SPECTROMETRY

Matrix-assisted laser desorption-ionization (MALDI)-postsource decay ( PSD) was used to differentiate glycoprotein-released N-linked oligosac charide isomers directly from aliquots of glycosidase digests and peak fractions collected from high-pH anion exchange chromatography (HPAEC ) with minimal sample handling and material. With the implementation o f instrumental tuning and acquisition controls, MALDI-PSD of NMR-chara cterized high-mannose, hybrid, and complex standards resulted in spect ra with reproducible fragment ion peak intensity ratios which correlat ed well to the respective oligosaccharide branching patterns, A ''know ledge-based'' strategy was utilized to characterize unknown isomeric N -glycan structures in which specific fragment ion types and their dist ributions in the unknown PSD spectrum were compared to those in PSD sp ectra of standards possessing similar structural features. This PSD kn owledge-based isomeric differentiation strategy was applied to disting uishing recombinant glycoprotein-derived Man7 D1 versus D2/D3 isomers directly from a PNGaseF digest aliquot of high-mannose N-glycans based on branching differences. A precursor ion selection device was employ ed to isolate the component of interest from the mass profile without additional chromatographic isolation steps. MALDI-MS signal-to-backgro und was maximized for direct PSD with on-the-probe sample clean-up met hods. The asialo complex N-glycan PSD knowledge base was used to diffe rentiate HPAEC peak fractions containing the tri- and tri'-antennary b ranching isomers and two tetraantennary isomers with antennal versus c ore fucose locations. Although the particular asialo complex N-glycan isomers here were well separated by HPAEC, MALDI-MS alerted us to thei r presence using mit-derived monosaccharide compositions and PSD fragm entation allowed us to differentiate these structures using the HPAEC elution positions as guides. (C) 1998 Academic Press.