Determining the Antennary Structure by Exoglycosidase
Sequencing
The 2-aminobenzoic acid (2-AB)-labeled glycans derived from R27T were
incubated overnight at 37°C with various exoglycosidase enzymes in 50 mM
sodium acetate pH 5.5. The detailed information for N-glycan release and
labeling was described in the Experimental section in the Supporting
Information. Enzymes were removed by a protein-binding membrane, and
samples were analyzed by HILIC-HPLC using an LSN2-40m-Nlink-35%_G100
and a LudgerSep-N2 column (LS-N24.6×150) on a Waters 2795 HPLC
instrument linked to a 2475 fluorescence detector controlled by Empower
software version 2, build 2154. The detailed HPLC condition was
described in the Experimental section in the Supporting Information.
2-AB-labeled glucose homopolymer (Ludger product CAB-GHP-30, 2-AB
glucose homopolymer ladder) was used as a system suitability standard as
well as an external calibration standard for GU allocation (the system
is deemed to be within specifications if the peak width at half height
for GU10 is less than 0.4 min).