Multimode High-Performance Liquid Chromatography of Fluorescently Labeled Oligosaccharides from Glycoproteins

Abstract

A polymeric, secondary amine stationary phase was used to develop a method for the separation of 2-aminobenzamide (AB)-labeled neutral and anionic oligosaccharides from glycoproteins. Sequential hydrophilic interaction liquid and anion exchange chromatography were performed in the same chromatographic analysis (multimode HPLC) and unambiguous separation between neutral and anionic oligosaccharides was accomplished. Improved resolution was also achieved. The oligomannosidic-type structures from bovine ribonuclease B (Man4GlcNAc-AB–Man9GlcNAc-AB) were separated not only by size, but also by the branch location of terminal Manα(1 → 2)-linked residues. Sialylated lactosamine-type oligosaccharides from human α1acid glycoprotein were resolved according to charge, Fuc content, and the number of Galβ(1 → 4)Galβ(1 → 3) repeats. The minor fucosylated and polylactosamine species were well separated from the major sialylated tetra-antennary oligosaccharides. Volatile mobile phases were used to minimize sample handling for matrix assisted laser desorption mass spectrometric analysis of peak fractions. Novel polylactosamine structures (3–5 repeats) from α1-acid glycoprotein were deduced from the molecular weight analysis. Multimode HPLC should prove useful for preparing low pmol quantities of fluorescently labeled oligosaccharides with fewer steps and minimal sample handling for mass spectrometric analysis, important requisites for structural studies of sample-limited glycoproteins.