Glycan reductive amino acid coded affinity tagging (GRACAT) for highly specific analysis of N-glycome by mass spectrometry.

N-glycans are involved in a variety of biological processes and associated with many diseases. However, the sensitive and specific detection of glycans via mass spectrometry (MS) remains challenging due to their relatively low abundance and low ionization efficiency. In this work, a new method termed glycan reductive amino acid coded affinity tagging (GRACAT) is developed to address these problems. Through labeling the reducing ends of glycan with a biotinylated arginine, the ionization efficiency of glycan was increased nearly 50-fold and glycan isomers were discriminated by improved signals of fragments in tandem spectra. Moreover, the strong affinity interaction between streptavidin and biotin enabled highly specific enrichment of the biotin-tagged glycans from the mixture of proteins and peptides. We successfully applied GRACAT in the profiling of N-glycome in human serum and bovine milk, in which a total of 55 and 67 N-glycans were detected respectively.