Site-Specific Profiling of Serum Glycoproteins Using N-Linked Glycan and Glycosite Analysis Revealing Atypical <i>N</i>-Glycosylation Sites on Albumin and α‑1B-Glycoprotein

Most serum proteins are N-linked glycosylated, and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum N-glycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by at least two glycans, with a majority of them being sialylated. Specifically, 3/4 of glycosites were modified by biantennary N-glycans and 1/3 of glycosites were modified by triantennary sialylated N-glycans. In addition, two novel atypical glycosites (with N–X–V motif) were identified and validated from albumin and α-1B-glycoprotein. The widespread presence of these two glycosites among individuals was further confirmed by individual serum analyses.