Structural characterization of glycoproteins remains among the most challenging areas of glycomics due to the requirement of large quantities of samples and laborious biochemical steps involved in the analytical procedure. Here we report the structural characterization of glycoproteins separated on a 2-D gel by using a MALDI-QIT-TOF MS where QIT is quadrupole IT. The combination of MALDI-ion source and QIT-appears to generate a unique tendency to cause fragmentation of glycopeptides without collision-induced dissociation. The majority of such fragmentations observed in our study result from the cleavage of sugar linkages, but not of peptide-peptide or peptide-sugar linkages. This unique feature allows us to perform pseudo-MS3 analysis of a fragmented glycopeptide. A small gel spot of a glycoprotein in the abundance range of low picomoles was enough for the mass spectrometer to analyze fragmentation pathway of the sugar linkage and peptide backbone. In this study, we demonstrate direct determination of glycosylation sites and Winked glycan-sequences of the tryptic glycopeptides of Drosophila glycoproteins. Glycopeptides with various IMWs up to similar to 4000 Da were suitable for structural analysis, including its attachment site and the amino acid sequence, of the glycopeptide through multistage mass spectrometric analysis.