Liquid chromatography-electrospray mass spectrometry was utilized to analyze peptide mapping of a glycoprotein ribonuclease B to obtain its primary structure. The glycosylated site was determined by comparison of peptide mapping before and after glycanase treatment.Tandem MS(MS/MS)was performed to analyze the structure of N-linked glycan and deglycosylated peptide. The nature of glycan was determined to be of highmannose type by mass spectrometry after the treatment with alpha-mannosidase. In addition the relative abundance of heterogeneous glycopeptides was quantified. This method is rapid and sensitive for the characterization of glycoproteins with N-linked glycan.