Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search.

Large-scale identification of -linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of -linked intact glycopeptides from -linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the -linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched -linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate -linked deglycopeptides. Both -linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From -linked deglycopeptides data sets, 764 -linked glycoproteins, 1699 -linked glycosites and 3328 unique -linked deglycopeptides were identified. Four types of -linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the -linked deglycopeptides. The spectra of these -linked deglycopeptides were utilized for -linked deglycopeptides library construction and identification of -linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of -linked intact glycopeptides. In total, 526 -linked glycoproteins, 1036 -linked glycosites, 22,677 -linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at -linked intact glycopeptide level.