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.