Confident characterization of the microheterogeneity of protein glycosylation 
through identification of intact glycopeptides remains one of the toughest 
analytical challenges for glycoproteomics. Recently proposed mass spectrometry 
(MS)-based methods still have some defects such as lack of the false discovery 
rate (FDR) analysis for the glycan identification and lack of sufficient 
fragmentation information for the peptide identification. Here we proposed 
pGlyco, a novel pipeline for the identification of intact glycopeptides by using 
complementary MS techniques: 1) HCD-MS/MS followed by product-dependent 
CID-MS/MS was used to provide complementary fragments to identify the glycans, 
and a novel target-decoy method was developed to estimate the false discovery 
rate of the glycan identification; 2) data-dependent acquisition of MS3 for some 
most intense peaks of HCD-MS/MS was used to provide fragments to identify the 
peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact 
glycopeptides could be confidently identified. With pGlyco, a standard 
glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant 
intact glycopeptides were identified with detailed spectral information of both 
glycans and peptides.