Determining N-glycan Structures by WAX/HILIC-HPLC with Fluorescence Labeling of Glycans
The 2-AB-labeled glycans derived from R27T were separated using a LudgerSep-C3 WAX-HPLC column with manual fraction collection of the separated peaks. Collected fractions were desalted by evaporation of the HPLC buffer in a centrifugal evaporator followed by the addition of 1 mL aliquots of water to each fraction and subsequent centrifugal evaporation of the water. This process was repeated until no salt deposit was visible. Half of each fraction was analyzed directly by LudgerSep-N2 HILIC-HPLC, and the remaining half was digested with sialidase then analyzed by LudgerSep-N2 HILIC-HPLC. For sialidase digestion, aliquots of 2-AB-labeled glycan fractions were incubated overnight at 37°C with Sialidase (E-S001; QABio) (a368S; specific for a2-3, -6, -8, and -9 sialic acids) in 50 mM sodium acetate pH 5.5. Enzyme was removed using a protein-binding membrane before analysis by LudgerSep-N2 HILIC-HPLC.