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  • Title: Isomeric differentiation of asparagine-linked oligosaccharides by matrix-assisted laser desorption-ionization postsource decay time-of-flight mass spectrometry.
    Author: Rouse JC, Strang AM, Yu W, Vath JE.
    Journal: Anal Biochem; 1998 Feb 01; 256(1):33-46. PubMed ID: 9466795.
    Abstract:
    Matrix-assisted laser desorption-ionization (MALDI)-postsource decay (PSD) was used to differentiate glycoprotein-released N-linked oligosaccharide 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 of instrumental tuning and acquisition controls, MALDI-PSD of NMR-characterized high-mannose, hybrid, and complex standards resulted in spectra with reproducible fragment ion peak intensity ratios which correlated well to the respective oligosaccharide branching patterns. A "knowledge-based" strategy was utilized to characterize unknown isomeric N-glycan structures in which specific fragment ion types and their distributions in the unknown PSD spectrum were compared to those in PSD spectra of standards possessing similar structural features. This PSD knowledge-based isomeric differentiation strategy was applied to distinguishing 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 employed to isolate the component of interest from the mass profile without additional chromatographic isolation steps. MALDI-MS signal-to-background was maximized for direct PSD with on-the-probe sample clean-up methods. The asialo complex N-glycan PSD knowledge base was used to differentiate HPAEC peak fractions containing the tri- and tri'-antennary branching isomers and two tetraantennary isomers with antennal versus core fucose locations. Although the particular asialo complex N-glycan isomers here were well separated by HPAEC, MALDI-MS alerted us to their presence using m/z-derived monosaccharide compositions and PSD fragmentation allowed us to differentiate these structures using the HPAEC elution positions as guides.
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