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).