De novo determination of fucose linkages in N-glycans and the unusual N-glycans in bee venom

N-linked glycosylation are one of the most important post-translational modification of proteins. Fucosylated N-glycans are related to many biological processes and they are commonly used as biomarkers. However, determining fucose linkages in N-glycans remains challenging, and most of the fucose linkages, particularly the linkages in the antenna of N-glycans remain unidentified. In this work, a simple multi-stage tandem mass spectrometry method based on dissociation mechanisms was developed for the de novo determination of fucose linkages in N-glycans. Using collision induced dissociation (CID), fucose linkages in intact N-glycans (i.e., without permethylation, reduction, or other derivatization) extracted from pine nuts and human milk were identified. We demonstrate that fucose linkages in N-glycans, including the 1-3 linkage in the core of N-glycans, the 1-6 linkage in the core of N-glycans, the linkage in the (13) antenna of biantennary N-glycans, and the linkage in the (16) antenna of biantennary N-glycans, were identified through CID spectra. The procedure does not require the N-glycan standards, and the entire structural determination process is summarized in a flowchart for automation. Applications of this method to N-glycans extracted from bee venom led to the discovery of two unusual N-glycans, which are not expected to exist according to the current biosynthetic pathways.