N-glycans released from glycoproteins using a commercial kit and comprehensively analyzed with a hypothetical database

摘要

The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities. This work describes a strategy that combines an efficient release, labeling and liquid chromatography-mass spectral analysis with the use of a comprehensive database to analyze N -glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry (MS) analysis and fluorescence detection of N -glycans. A hypothetical database, constructed using GlycResoft, provides all compositional possibilities of N -glycans based on the common sugar residues found in N -glycans. In the initial version this database contains 8,700 N -glycans, and is compatible with MS instrument software and expandable. N -glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than had been previously reported. This strategy was then used to analyze the N -glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N -glycan data from those cells was obtained efficiently and their differences compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N -glycan analysis.