Glycosylation is one of the most common and complex post-translational modifications in biological systems.Information on glycan heterogeneity is increasingly recognized as crucial to understanding glycoprotein structure and function.Due to the cost-saving of nonspecific enzymes such as pronase E,several research groups have used them for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins.However,several glycopeptides with different amino acid residuals that share the same glycan structure could display lower detection sensitivity and lead to complicated mass interpretation using this method.In our study,increasing the enzyme/glycoprotein ratio and prolonging the digestion time were used to overcome those disadvantages.In addition,a permanent charge derivatization reagents,TMPP-Ac-OSu,was also employed for the determination of N-linked oligosaccharides.The experimental strategy was validated using glycoproteins with known oligosaccharide structure,Ribonuclease B(high mannose type),Ovalbumin(hybrid type)and Transferrin(complex type).