Analysis of major complex fucosylated N-glycans of hESC. A. The major sialylated N-glycan signal with complex fucosylation feature (S1H5N4F2) was subjected to exoglycosidase sequencing with linkage-specific fucosidases and broad-range sialidase. As outlined in the reaction scheme, the signal was shown to be an approximately 1:1 mixture of α1,2- and α1,3-/α1,4-fucosylated biantennary complex-type N-glycans with core fucosylation. B. and C. The acidic N-glycan fraction was permethylated and subjected to MS/MS fragmentation. Both S1H5N4F2 (B.) and S1H5N4F1 (C.) produced fragments that supported the structure assignments as indicated in the schematic drawings. D. The studied hESC lines were shown to express four of the known human fucosyltransferases. Minimal glycan acceptor and product specificities for the encoded enzymes are shown in the schematic drawing.