N-linked glycoproteome profiling of human serum using tandem enrichment and multiple fraction concatenation

N-linked glycosylation is an important protein posttranslational modification that is involved in numerous biological processes. Different methods, including chemical reaction and affinity interaction, have been developed to enrich glycosylated peptides or proteins from biological systems. However, due to the common occurrence of low glycosites occupancy in proteins and the low efficiency of enrichment approaches, only a small fraction of protein glycosites have been reported. In this study, we combined the glycopeptide enrichment strategy for broad analysis of human serum N-glycoproteins using a tandem enrichment method coupling lectin affinity capture with HILIC. This strategy was applied to profile the human serum N-linked glycoproteome, and it resulted in 32 and 14% more N-glycosites than could be identified with the common lectin affinity capture or HILIC approaches, respectively. With an additional dimension of glycopeptides separation using high-pH reversed phase liquid chromatography or off-gel electrophoresis, the number of identified glycosites was increased by 3.1-fold and 1.8-fold, respectively. These results demonstrate that tandem enrichment methods, especially when followed by high-pH reversed-phase prefractionation, can greatly improve the power of N-glycoproteome analysis. In total, 615 N-glycosites from 312 glycoproteins (protein group) were mapped using high-accuracy mass spectrometry.