Protein glycosylation may regulate protein functions,affect the turnover,antibody dependent cell-mediated cytotoxicity of biopharmaceuticals.Characterization of glycan structure has been hindered by the technical challenges caused by the non-template synthesis of glycans,low abundance of modified glycoforms and its huge micro heterogeneity.Here we demonstrate the enrichment and identification of glycosylated protein,both at the global level or site-specific mode.We showed that by synthesizing pH-sensitive or temperature-sensitive reagent,the sensitivity on the enrichment and detection of glycoprotein could be improved significantly.For the site-specific characterization of the glycosylation of proteins,an integrated strategy has been developed,which consists of 2-dimensional electrophoresis (2-DE) separation of the protein isoforms from different charge species,in-gel extraction of intact glycopeptides,and a novel strategy for interpretation of mass spectrometric spectra from intact glycopeptides.We illustrated the detailed structure of site-specific glycans and their dynamic change along with the series of 2-DE spots for a proteins with 7 N-glycosites,which provided a solid pipline for the quality control of complex glycoprotein drugs.The strategy was also applied on exploring the glycosylation status of the more complicated glycoproteins,EGFR in biosystem.